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REGIONA 

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S.   DEPARTMENT  OF  AGRIC 

BUREAU  OF  ANIMAL  INDUSTRY.— BULLETIN  N 
A.   D.  MELVIN,  Chief  of  Bureau. 


SOUTHERN  BRANCH, 

UNIVERSITY  OF  CALIFORNIA, 

LIRRA 


i  in  Cheese  ripening 


CAMEMBERT  AND  ROQUEFORT. 


BY 


CHARLES  THOM,   Ph.  D.. 

Mycologist  in  Cheese  Investigations,  Dairy  Division,  Bureau  of 
Animal  Industry. 


^nos 
:sj9Aiun. 


WASHINGTON: 
GOVERNMENT   PRINTING   OF 

[906; 


11 K  BIKEAl    OF  ANIMAL  IN 


^vin,  D.  V.  S 


int  Chief:  A.  M.  Fakkinu  ion.  15.  S.,  1).  V.  M. 
'Clerk:  E. 


B.Jonbs,  LL.  M..M.  1). 
,ery  Division:  Ed.  II.  Webster,  M.  §,,  ehietj  Clarence  15.  Lank,  15.  S.,  assistant  chief. 
aspection  Division:  Kick  P,  Steddom,  V.  S.,  chief. 
Quarantine  Division:   RiuiAisn  W.  Hickman,  I'll,  (i.,  V.  M.  1).,  chief. 
Animal  Husbandman:  George  M.  Rommel,  B.  S.  A. 
Editor:  James  M.  Pickens. 
Artist:  W.S. 1).  IIainks. 
Librarian:  Beatrice  C.  Obekly. 


LABORATORIES. 

Biochemic  Division:  Marion  Dorset,  M.  D.,  chief. 

Pathological  Division:  John   K.  Moiileh,  A.  M.,  V.  M.  D.,' chief. 

Zoological  Division:  Brayton  II.  Ransom,  B.  Sc.,  A.  M.,  scientiQc  assistant  in  char; 

EXPERIMENT    STATION. 

Superintendent:  E.  C.  SchroeDer,  M.  1).  V.;  expert  assistant,  W.  E.  Cotton. 

MEAT    INSPECTION. 

Inspectors  in  charge. 


Austin,  Minn.— Dr.  M.  O.  Anderson,  care  George 
A.  Hofmel  &  Co. 

Baltimore.  Md.     Dr.  II.  A.  Hedrick,  215  St.  Paul 
street. 

Bloomington,  111.— Dr.  Frederick  Braginton,  care 
Continental  Packing  Company. 

Boston,  Mass.— Dr.  J.  P.  Ryder,  Ml  Milk  street. 

Brightwood,    Mass.     Dr.     \Y.    J.    Murphy,    care 
Springfield  Provision  Company. 

Buffalo,  N.  V.  -Dr.  B.  P.  Wcnde.  Livestock  Ex- 
change Padding,  East  Buffalo. 

Pj.pids,  Iowa.— Dr.  T.  A.  Shipley,  care  T.  M. 
Piiioialr  &  Co. 

Chicago,  111.— Dr.  S.  E.  Bennett,  room  316   Ex- 
change Building,  Union  Stock  Yards. 

Cincinnati,  Ohio.— Dr.  A.  G.  G.  Richardson,  care 
Union  Stock  Yards. 

Cleveland,  Ohio.— Dr.  E.  P.  Schaffter,  care  Cleve- 
land Provision  Company. 

Davenport,    Iowa.— Dr.    E.    L.    Bertram,    caic 
Henry  Kohrs  Packing  Company. 

Denver,  Colo.     Dr.    \Y.    E.    Howe,  care    Western 
Packing  Company. 

Des  Moines,  Iowa.— Dr.  A.  B.  Morse,  care  The 
Agar  Packing  Company. 

Detroit,  Mich.— Dr.  L.  K.  Green,  care  Hammond, 
Standish  &  Co. 

Eau  Claire,  Wis.— Dr.  G.  W.  Butler,  care  Drum- 
mond  Brothers. 

Fort  Worth,  Tex.— Dr.  A.  II.  Wallace,  care  Swift 
&  Co. 

Hutchinson,  Kans. — Dr.   J.    E.    Blaekwell,   care 
Hutchinson  Packing  Company. 
■"Mwiapolis,  Ind. — Dr.  N.  C.  Sorensen,  care  Kin- 
"o. 

V.  J. — Dr.  Julius  Huelsen,  care  The 
"*ek  Yard  Company. 

-Dr.  L.  R.  Baker,  room  338 

E.  Rishel,  care  Cudahy 

"■orge,  507  Johnson 

,  care  Macbeth  & 

/'.   F.  Price,  care 

-are  Jacob  E. 


Milwaukee,  Wis.— Dr.  A.   E.    Behnke,  room   432 

Federal  Building. 
Nashville,  Term. — Dr.  W.  B.   Lincoln,  can 

nessee  Packing  and  Provision  Company. 
National  Stock  Yards,  111. -Dr.  .1.  B.  Clancy. 
Xelira  ska  City,  Xebr.-  Dr.  W.  II.  Gibbs,care  Mor- 

ton-Gregson  Company. 
Newark,  N.  J.— Dr.  Thomas  Castor,  care  Swift  & 

Co.,  Harrison  Station. 
New  Haven,  Conn.— Dr.  Albert  Long,  care  Sperry 

A;  Barnes. 
New  York,  N.  Y.— Dr.  II.  N.  Waller,  109  West 

Forty-second  street. 
Ottumwa,  Iowa.— Dr.  Joshua  Miller,  care  John 

Morrell  &  Co. 
Philadelphia,  Pa.— Dr.  C,  A.  Schaufler,  134  South 

Seconcl  street. 
Pittsburg,    Pa.— Dr.    F.    W.    Ainsworth,    Union 

Stock  Yards. 
Portland,  Oreg.  — Dr.  Clarence  Loveberry,  room 

402,  custom  house  (new). 
Quincy.HL— Dr.  J.S.  'Kelly,  care  BlomerA  Michael 

Co. 
St.  Louis,  Mo.— Dr.  J.  J.  Brougham,  care  Missouri 

stock  Yards  Company. 
San  Diego,  Cal.— Dr.  Robert  Darling,  care  Charles 

S.  Hard)-. 
San  Francisco,  Cal.— Dr.  George  S.  Baker,  Sixth 

and  Townsend  streets. 
Seattle,  Wash.— Dr.  O.  B.  Hess,  care  Frye-Bruhn 

Company. 
Sioux  City,  Iowa.— Dr.  G.  A.  Johnson,  Exchange 
.  Building. 
South   Omaha,  Nebr.— Dr.  Don  C.   Ayer,   Post- 

Office  Building. 
South  St.  Joseph,  Mo.— Dr.  George  Ditcwig. 
South  St.  Paul,  Minn— Dr.  K.  I).  Ketchum. 
Tacoma,  Wash.— Dr.  E.  C.  Joss,  care  Carstc 

Packing  Company. 
Topcka,    Kans.— Dr.  F.  L.  DeWolf,  care  Char 

Wolfl  Packing  Company. 
Waterloo,  Iowa.— Dr.  T.  W.  Scott,  care  The  Rf 

Packing  Company. 
Wichita,  Kans.— Dr.  W.  N.  Neil,  care  John  C 

ahv  Company. 
Worcester,  Mass.— Dr.  E.  P.  Dowd,  care  Wl 


Pevey  &  Dexter  Co. 


U.  S.   DEPARTMENT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY.— Bulletin  No.  82. 

A.  D.  MELVIN,  Chief  of  Bureau.    . 


FUNGI   IN   CHEESE   RIPENING 

CAMEMBERT  AND  ROQUEFORT. 


BY 


CHARLES  THOM.   Ph.  D., 

Mycologist  in  Cheese  Investigations ,  Dairy  Division,  Bureau  of 
Animal  Industry. 


WASHINGTON: 

GOVERNMENT   PRINTING   OFFICE. 

1906. 


LETTER  OF  TRANSMITTAL. 


U.  S.  Department  of  Agriculture, 

Bureau  of  Animal  Industry, 

Washington,  D.  C,  February  6,  1906. 
Sir:  I  have  the  honor  to  transmit  herewith  the  manuscript  of  an 
article  entitled  "Fungi  in  Cheese  Ripening:  Camembert  and  Roque- 
fort," by  Charles  Thom,  Ph.  D.,  and  to  recommend  its  publication  as 
Bulletin  No.  82  of  the  series  of  this  Bureau.  This  is  the  second  paper 
dealing  with  the  cooperative  experiments  in  soft -cheese  making 
undertaken  by  the  Dairy  Division  of  this  Bureau  in  conjunction  with 
the  Storrs  (Conn.)  Agricultural  Experiment  Station,  the  first  paper 
having  been  published  as  Bulletin  No.  71  of  this  Bureau. 

These  experiments  have  been  carried  on  at  the  Storrs  Station  under 
the  general  direction  of  Prof.  L.  A.  Clinton,  the  station  director,  and 
under  the  personal  supervision  of  Dr.  H.  W.  Conn,  the  station  bac^ 
teriologist,  in  accordance  with  the  plan  outlined  in  the  introduction 
to  Bulletin  No.  71. 

While  there  are  many  problems'yet  to  be  investigated  with  refer- 
ence to  the  manufacture  in  this  country  of  soft  cheeses  of  the  best 
European  types,  this  article  indicates  that  good  headway  is  being 
made  in  that  direction,  and  it  is  believed  that  the  information  here 
presented  is  of  considerable  scientific  and  economic  value. 
Respectfully, 

A.  D.  Melvin, 
Chief  of  Bureau . 
Hon.  James  Wilson, 

Secretary  of  Agriculture. 
2 


CONTENTS 


Page. 

Introduction i 5 

Camembert  cheese 5 

Resume"  of  previous  paper 5 

Culture  media  and  methods 6 

Effect  of  a  fungus  upon  a  culture  medium 8 

Literature  of  cheese  fungi 8 

Biological  analysis  of  a  cheese 9 

The  flora  of  Camembert  cheese .". 10 

Outline  of  the  work 11 

Relation  of  molds  to  acidity 12 

The  breaking  down  of  casein 14 

Liquefaction  of  gelatin 15 

Raulin  's  fluid 16 

Casein 16 

Sterile  milk  and  curd 17 

Does  the  mycelium  penetrate  the  cheese  1 17 

Camembert  Penicillium  upon  cheese 18 

Comparative  studies  of  fungous  digestion 18 

Flavors 21 

Temperature c 23 

Humidity 24 

Inoculating  material 25 

Inoculation  with  Penicillium : . . .  26 

Vitality  of  spores 27 

Contaminations 27 

Roquefort  cheese 28 

Cheeses  related  to  Roquefort 29 

American  Brie  and  Isigny 30 

Molds  referred  to  in  this  paper 31 

The  Camembert  mold  (Penicillium  camemberti) 32 

Technical  characterization  of  the  Camembert  mold 33 

The  Roquefort  mold  (Penicillium  roqueforti) 34 

Technical  characterization  of  the  Roquefort  mold 35 

Oidium  lactis 36 

Summary 38 

Camembert  cheese 38 

Roquefort  cheese 38 

Other  varieties  of  .cheese 39 

Bibliography 40 


ILLUSTRATION'S 


Fio.  1.  Camembert  Penicillium  (P.  camemberti) 32 

2.  Roquefort  Penicillium  (P.  roqueforti ) 35 

3.  Oidium  lactis 37 

3 


Digitized  by  the  Internet  Archive 

in  2007  with  funding  from 

Microsoft  Corporation 


http://www.archive.org/details/fungiincheeseOOthomiala 


FUNGI  IN  CHEESE  RIPENING:  CAMEMBERT  AND 

ROQUEFORT. 


INTRODUCTION. 

It  has  been  shown  in  a  previous  bulletin  that  certain  fungi  are  the 
active  agents  indispensable  to  the  ripening  of  Camembert  cheese. 
The  general  results  and  the  data  upon  which  they  rest  are  there  dis- 
cussed, but  the  more  special  mycological  studies,  involving  several 
lines  of  work,  remained  to  be  brought  out  in  greater  detail.  These  fall 
naturally  under  two  heads:  (1)  The  physiological  studies  of  the  func- 
tions of  particular  species  in  the  ripening  processes  of  Camembert, 
Roquefort,  and  certain  related  types  of  cheese;  (2)  the  classification 
and  description  of  these  and  other  forms  occurring  in  dairy  work. 
This  paper  includes  only  the  work  done  under  the  first  head.  The 
description  of  the  fungi  occurring  in  dairy  work  is  reserved  for  another 
paper. 

Aside  from  such  obligations  as  are  mentioned  in  the  discussion  of 
special  topics,  the  author  wishes  to  acknowledge  the  assistance  of  Dr. 
B.  B.  Turner,  Prof.  W.  A.  Stocking,  Mr.  A.  W.  Bosworth,  and  Mr. 
T.  W.  Issajeff,  members  of  the  experiment  station  staff,  in  numerous 
cases  where  the  work  of  each  presupposes  the  results  of  the  other,,  and 
especially  to  acknowledge  the  constant  assistance  of  the  supervisor  of 
the  investigation,  Dr.  H.  W.  Conn,  with  whom  the  cheese  problems 
have  been  fully  discussed  at  every  stage. 

CAMEMBERT  CHEESE. 
RESUME    OF   PREVIOUS    PAPER. 

The  biological  conditions  and  the  physical  changes  encountered  in 
the  production  of  a  Camembert  cheese  from  market  milk  may  be 
restated  from  our  former  bulletin1"  as  a  basis  for  defining  the  special 
problems  of  the  mycologist. 

Milk  as  ordinarily  received  contains  bacteria  of  many  species  and 
the  germinating  spores  of  numerous  fungi  from  the  stable  and  from 
the  food  of  the  cattle.     When  such  milk  is  curdled  for  cheese  making, 

aTlie  figure  references  tin-  to  bibliography  at  end  of  bulletin. 


6  •      FUNGI    IN    CHEESE    RIPENING. 

representatives  of  all  of  these  species  are  inclosed  in  the  mass  of  coagu- 
him.  Freshly  made  cheese  from  this  curd,  then,  may  contain  any 
species  of  mold  or  bacterium  found  in  the  locality  which  is  capable  of 
living  in  milk  or  its  products.  The  first  step  in  the  ripening  of  a  Ca- 
me nibert  cheese  is  the  production  of  lactic  acid.  The  lactic  bacteria 
very  soon  increase  their  rate  of  multiplication  so  enormously  as  to  be- 
come entirely  dominant.  The  acid  produced  by  these  forms  soon 
reaches  a  percentage  sufficiently  high  to  restrict  the  further  growth 
of  nearly  every  other  species  of  bacteria,  and  even  to  eliminate  the 
organisms  themselves.  In  a  time  varying  from  a  few  hours  to  three 
or  four  days,  according  to  the  proportional  numbers  of  these  antago- 
nistic species  at  the  start,  further  bacterial  growth  seems  to  be  entirely 
stopped.  Bacterial  development  can  not  begin  again  until  this  acidity 
is  reduced  below  the  critical  point  for  the  species  involved,  and  even 
then,  since  the  acid  is  neutralized  on  the  outside  first,  for  most  species 
it  begins  at  the  surface  and  works  slowly  inward.  The  uncertainties 
due  to  the  presence  of  many  species  of  bacteria  in  the  milk  are  in  this 
way  avoided  by  the  natural,  simple,  and  almost  universally  successful 
process  of  souring. 

The  further  ripening  of  a  Camembert  cheese  is  attended  by  a 
gradual  reduction  of  this  acidity  until  the  ripe  cheese  is  usually  alka- 
line to  litmus.  At  the  same  time  the  mold  action  in  the  mass  of  curd 
produces  chemical  changes  which  in  from  three  to  five  weeks  reduce 
the  previously  insoluble  mass  to  a  high  percentage  of  solubility  in 
water.  In  the  later  stages  of  this  breaking  down  compounds  are 
formed  which  give  the  characteristic  odors  and  flavors  to  this  type  of 
cheese.  Associated  with  these  chemical  changes  there  is  a  progressive 
physical  change  from  the  firm  curd  to  a  soft,  buttery,  or  even  semi- 
liquid  texture,  characteristic  of  ripe  cheese.  The  biological  problems 
then  were,  in  general,  the  determination  of  what  organisms  cause — 

(1)  The  changes  in  the  acidity  of  the  curd. 

(2)  The  breaking  down  of  the  casein,  with  the  associated  changes  in 
the  physical  character  of  the  cheese. 

(3)  The  production  of  the  flavors. 

(4)  The  recognition  and  control  of  deleterious  species. 

CULTURE  MEDIA  AND  METHODS. 

The  common  dairy  fungi  grow  readily  upon  any  of  the  standard  cul- 
ture media.  Among  the  media  used  have  been  peptone  agar,  whey 
gelatin,  sugar  gelatin  with  or  without  the  addition  of  litmus,  milk 
agar,  gelatin  and  agar  made  with  Raulin's  fluid,  potato  agar,  potato 
plugs,  and  sterilized  milk  and  curd.  Special  studies  have  involved 
other  preparations.  The  fact  that  these  fungi  grow  readily  upon  all 
the  common  media  has  led  to  the  selection  of  two  preparations  for  con- 
stant use,  and  the  careful  study  upon  these  of  all  species  found.     For 


CAMEMBERT   AND   ROQUEFORT.  7 

this  purpose  the  sugar  gelatin,  described  by  Conn2  for  the  qualitative 
bacteriological  analysis  of  milk,  and  potato  agar  have  been  used. 

The  sugar-gelatin  formula  produces  an  accurately  titrated  medium 
in  which  every  effort  is  made  to  secure  a  uniform  composition. 
Although  absolute  uniformity  in  chemical  and  physical  properties  is 
never  obtained,  the  reaction  of  many  species  of  fungi,  when  grown 
upon  successive  lots  of  gelatin  made  after  this  formula,  have  been  so 
reliable  as  to  commend  its  use  for  determining  physiological  charac- 
ters. It  seems  clearly  shown,  therefore,  that  slight  variations  in  the 
composition  of  the  medium  do  not  produce  great  differences  in  the 
species  studied  in  this  paper.  In  the  discussion  of  the  relation  of  a 
mold  to  this  gelatin  it  must  be  borne  in  mind  that  the  same  results 
might  not  follow  the  use  of  any  other  formula. 

The  other  medium,  the  potato  agar,  was  selected  because  of  its  use 
in  many  mycological  laboratories.  In  this  medium  uniform  compo- 
sition can  hardly  be  claimed.  The  following  process  has  been  used  in 
this  work:  The  potatoes  are  carefully  washed,  pared,  and  sliced,  then 
slowly  heated  for  about  two  hours  in  approximately  two  volumes  of 
water.  At  the  close  of  the  heating  the  water  is  allowed  to  boil.  The 
whole  is  then  filtered  through  cloth,  and  commonly  through  cotton 
also,  water  being  added  to  make  up  the  losses  of  evaporation  and  fil- 
tering. To  this  is  added  1  per  cent  of  shredded  agar.  It  is  then 
heated  for  from  twenty  to  thirty  minutes  in  the  autoclave  to  120°  C. 
or  higher,  when  it  may  at  once  be  put  into  tubes  for  use,  or,  if 
cloudy,  it  may  be  very  quickly  filtered  through  absorbent  cotton, 
after  which  it  should  be  quite  clear.  The  uncertainties  in  the  com- 
position of  this  medium  result  from  the  differences  in  the  potato  ex- 
tract itself  and  from  the  fact  that  the  difficulties  in  filtering  this 
extract  take  out  a  varying  amount,  which  is  replaced  with  water. 
Titration  shows  that  this  medium  is  nearly  neutral  (4-6  acid  on  Ful- 
ler's scale)  in  cases  tested  to  phenolphthalein;  consequently  it  is  used 
without  neutralizing.  Culture  and  study  of  the  same  species  upon 
successive  lots  of  this  medium  show  that  these  differences  in  compo- 
sition have  little  if  any  effect  upon  the  morphology  of  the  species 
studied. 

P6tri-dish  cultures  have  been  used  continually  because  they  admit 
of  direct  study  under  the  microscope.  Slanted  test  tubes  were  found 
useful  for  stock  cultures  and  for  gross  studies  of  physiological  effects, 
but  they  are  of  little  value  for  comparative  work.  It  is  useless  to 
attempt  to  get  a  correct  idea  of  the  normal  gross  structure  of  these 
molds  from  fluid  mounts.  The  extremely  delicate  hypha;  are  so  tan- 
gled in  such  preparations  as  to  give  but  very  little  idea  of  their  ordi- 
nary appearance,  while  the  chains  of  conidia  break  up  immediately 
when  placed  in  any  fluid.  Such  mounts  are  useful  and  necessary  to 
get  at  details  of  cell  structure  and  cell  relations,  but  in  comparative 


8  FUNGI    IN    CHEE8E    RIPENING. 

studies  of  species  of  such  a  genus  as  Penicillium  their  value  is  only  that 
of  a  useful  accessory.  The  primary  source  of  comparative  data  must 
be  direct  study  of  the  growing  colony,  undisturbed  upon  the  culture 
medium,  with  the  best  lenses  that  admit  of  such  use. 

This  method  of  study  recognizes  that  morphology  is  the  basis  of 
fungus  determination,  but  takes  into  consideration — 

(1)  That  morphology  must  not  only  include  the  minutest  details 
of  cell  structure  and  cell  relations  such  as  are  undisturbed  in  fluid 
mounts,  but  also  the  appearance  and  character  of  the  colony. 

(2)  That  the  morphology  of  the  colony — i.  e.,  the  size  of  conidio- 
phore  and  fructification,  relation  of  these  to  substratum,  appearance, 
and  relations  of  aerial  and  submerged  mycelium — is  different  upon 
various  substrata,  but  has  been  found  to  be  characteristic  for  each 
particular  substratum. 

(3)  That  a  description  of  morphology  to  be  of  value  must,  there- 
fore, specify  the  formula  of  the  medium  used  and  the  conditions. 

Dilution  cultures  have  been  necessary  usually  to  obtain  the  colonies 
pure,  but  the  direct  transfer  of  large  numbers  of  spores  upon  a  plati- 
num needle  to  the  surface  of  gelatine  or  agar  plates  which  have  been 
allowed  to  cool  has  been  found  to  give  equally  reliable  results,  and  to 
have  many  advantages  for  the  study  of  species  once  obtained  in  pure 
culture.  This  is  often  spoken  of  as  inoculation  of  cold-poured  plates. 
Litmus  solution  may  be  used  with  either  gelatin  or  agar,  and  gives 
striking  evidence  of  differences  in  species  and  the  rate  of  their  physio- 
logical action.  Bacterial  contamination  has  been  usually  restrained 
by  the  addition  of  from  2  to  4  drops  of  normal  lactic  acid  to  8  or 
10  c.  c.  of  medium. 

EFFECT   OF   A    FUNGUS    UPON   A    CULTURE    MEDIUM. 

In  studying  the  relation  of  a  fungus  to  a  culture  medium  we  find  (1) 
that  the  fungus  absorbs  food  from  the  surrounding  medium;  (2)  that 
it  may  secrete  or  excrete  substances  into  the  medium  which  may 
transform  its  chemical  composition  and  its  appearance.  The  amount 
of  food  absorded  by  the  fungus  is  small,  and  for  our  purposes  may  be 
practically  ignored,  but  the  changes  induced  by  indirect  action — 
secretions  from  the  mycelium — are  great  and  far-reaching.  To  this 
latter  group  belong  the  changes  in  acidity,  digestive  effects,  and  fla- 
vors produced  by  fungi. 

LITERATURE    OF    CHEESE    FUNGI. 

A  review  of  the  literature  at  the  outset  showed  that  no  work  on  the 
fungous  flora  of  the  various  types  of  soft  cheese  had  been  published  in 
English.  Epstein,3  at  Prague,  studied  the  ripening  of  Camembert  and 
Brie  cheeses.  He  attributes  the  breaking  down  of  the  curd  in  French 
Brie  to  the  action  of  Penicillium  album,  but  denies  the  participation 


CAMEMBERT    AND    ROQUEFORT.  9 

of  molds  in  the  ripening  of  Camembert.  Johan-Olsen,4  in  Sweden,  has 
published  a  brief  review  of  the  fungi  related  to  the  ripening  of  Gamme- 
lost,  barely  mentioning  work  done  upon  Camembert.  Constantin  and 
Ray,5  in  France,  have  described  the  appearance  upon  the  cheese  of  the 
species  of  Penicillium  involved  in  the  ripening  of  the  French  Brie. 
Roger,6  also  in  France,  has  attributed  a  single  phase  of  Camembert 
cheese  ripening  to  the  activity  of  Penicillium  candidum,  for  which  he 
gives  no  description.  Of  these  references,  that  of  Epstein  and  that  of 
Constantin  and  Ray  describe  the  mold  found  upon  the  French  Brie 
sufficiently  clearly  to  aid  in  its  recognition.  A  popular  article,  signed 
Margaret,7  in  the  Creamery  Journal  of  October,  1904,  gives  in  entirely 
untechnical  language  a  very  satisfactory  description  of  the  appearance 
upon  cheese  of  the  penicillium  concerned  in  the  ripening  of  Camem- 
bert. The  general  insufficiency  of  the  literature  available  made  a 
first-hand  study  of  the  types  of  cheese  found  in  American  markets  the 
only  source  from  which  definite  information  could  be  secured. 

BIOLOGICAL   ANALYSIS    OF   A    CHEESE. 

In  the  biological  analysis  of  a  market  cheese  it  is  carefully  un- 
wrapped to  avoid  contamination  as  far  as  possible.  Series  of  dilution 
cultures  on  neutral  and  acid  media  are  made  at  once  from  each  part 
of  its  surface  which  shows  any  variation  in  appearance.  In  this  way 
all  the  surface  molds  and  bacteria  are  secured  in  one  set  of  plates. 
Afterwards  this  surface  is  examined  in  detail,  usually  with  a  lens,  the 
appearance  of  the  different  areas  being  noted,  and  direct  transfers 
from  each  area  made  to  cold  agar  or  gelatin  plates.  The  cheese  is 
then  cut  with  a  sterilized  scalpel  and  cultures  are  made  from  various 
portions  of  the  interior.  Usually  the  transfers  were  made  from  the 
center  and  from  the  area  just  inside  the  rind.  Any  part  showing  spe- 
cial appearances  is  reserved  for  a  separate  series  of  cultures. 

Most  of  the  brands  of  Camembert  cheese  found  in  our  markets,  as 
well  as  some  sent  by  Roger,  have  been  examined  in  this  way.  For 
comparison,  similar  studies  have  been  made  from  several  specimens  of 
Roquefort  cheese  bought  in  different  markets,  and  from  individual 
specimens  of  Gorgonzola  and  Stilton.  Single  studies  for  molds  have 
been  made  from  Limburger,  Port  du  Salut,  Brinse,  and  from  several 
brands  of  prepared  cheese  found  in  the  market.  From  these  cultures 
all  species  of  bacteria  found  have  been  isolated  and  handed  over  to  the 
bacteriologists.  Each  variety  of  mold  occurring  upon  these  cheeses 
has  been  isolated  and  studied.  It  has  been  possible  in  this  way  to 
show  that  a  comparatively  small  number  of  species  characteristically 
occur  upon  soft  cheese.  Although  this  list  may  be  greatly  extended 
by  including  forms  which  are  occasionally  found,  it  is  rather  surpris- 
ing that  a  restricted  group  of  species  occurs  with  much  regularity  in 
studies  of  cheese  from  so  widely  different  countries. 
21156— No.  82—06 2 


10  FUNGI    IN    CHEESE    RIPENING. 

To  study  the  origin  and  distribution  of  these  molds  several  labora- 
tories and  cheese  factories  have  been  visited  and  cultures  taken.  Cor- 
respondents in  distant  States  have  kindly  sent  cultures  of  molds 
occurring  in  their  work.  Among  those  who  have  sent  material  are 
Dr.  C.  E.  Marshall,  Agricultural  College,  Mich. ;  Mr.  E.  G.  Hastings, 
Madison,  Wis.;  Prof.  F.  C.  Harrison,  Guelph,  Ontario;  Dr.  H.  A. 
Harding,  Geneva,  N.  Y.,  and  Prof.  P.  H.  Rolfs,  Miami,  Fla.  Thus,  in 
addition  to  a  large  number  of  cultures  from  the  dairy  laboratories  of 
the  stations  at  Storrs  and  at  Middle  town,  we  have  accumulated  from 
various  sources  a  considerable  number  of  species  representing  the 
characteristic  molds  occurring  in  dairy  work,  as  well  as  many  forms 
collected  in  the  field  and  from  laboratories  not  associated  with  dairy 
investigation. 

THE    FLORA    OF    CAMEMBERT    CHEESE. 

Although  a  considerable  variety  of  molds  appeared  in  cultures  from 
Camembert  cheeses,  a  list  of  possibly  twenty  species  would  include 
those  which  were  often  found.  Among  these  there  are  perhaps  six 
species  of  Penicillium,  two  or  three  of  Aspergillus,  Oidium  lactis,  Clado- 
sporium  herbarum,  one  or  two  of  Mucor,  one  or  more  of  Fusarium, 
Monilia  Candida,  and  two  species  perhaps  related  to  it,  with  the  inci- 
dental occurrence  of  Acrostalagmus  cinnabarinus,  a  Cephalosporium, 
various  species  of  Alternaria,  and  Stysanus.  Besides  these,  yeasts  in 
large  numbers  and  considerable  variety  are  found  in  many  cases. 

The  comparison  of  the  results  of  culture  with  comparative  studies 
of  the  surfaces  of  different  brands  of  cheese  showed  that  a  single  spe- 
cies of  Penicillium  was  present  upon  every  Camembert  cheese  exam- 
ined. In  partially  ripened  cheeses  this  mold  often  covered  the  larger 
part  of  the  surface.  We  shall  call  this  the  "Camembert  Penicillium" 
or  the  "  Camembert  mold."  This  species  develops  a  large  and  charac- 
teristic growth  of  aerial  mycelium  in  addition  to  a  densely  felted  mass 
of  threads  which  penetrate  the  surface  of  the  cheese  for  1  or  2  mm.  and 
largely  constitute  the  rind.  In  all  except  a  few  very  old  cheeses 
which  were  almost  covered  with  red  slime  of  bacterial  origin  it  was 
readily  seen  to  be  the  dominant  species  upon  the  surface. 

Similarly,  cultural  data  showed  Oidium  (Oospora)  lactis  to  be  abun- 
dant upon  every  brand  of  Camembert.  This  mold  is  practically  in- 
distinguishable upon  the  surface  by  its  characters,  except  under  very 
favorable  conditions,  and  at  best  its  recognition,  even  with  a  hand  lens, 
is  not  often  certain.  Mycelium  of  this  fungus  develops  only  in  very 
moist  substrata,  and  is  usually  entirely  submerged.  Only  part  of  its 
chains  of  conidia  even  rise  above  the  surface.  In  old  and  very  ripe 
cheese,  when  the  rind  is  covered  with  yeasts  and  bacteria,  it  is  often 
difficult  under  the  microscope  to  find  the  spores  of  Oidium.  In  such 
cases,  unless  one  is  familiar  with  the  peculiar  smell  associated  with  its 


CAMEMBEET   AND   EOQUEFORT.  11 

action,  he  must  depend  entirely  upon  the  culture  for  evidence  of  its 
presence. 

No  other  species  of  mold  has  been  found  upon  every  cheese  exam- 
ined, although  no  market  cheese  has  failed  to  show  contamination 
with  at  least  one  or  two  of  the  other  fungi  listed  above.  In  other 
words,  comparative  biological  examination  of  imported  Camembert 
cheeses  established  the  fact  that  these  two  species  of  mold  were  pres- 
ent upon  them  all,  however  abundantly  they  might  be  contaminated 
with  other  forms.  The  examination  of  hundreds  of  cheeses  in  the  city 
markets  has  shown  the  presence  of  the  same  two  molds  upon  all  the 
brands  of  Camembert  offered  for  sale.  Such  analyses  clearly  estab- 
lished the  presence  of  these  molds  upon  the  ripe  cheese,  but  gave  no 
information  either  as  to  whether  they  were  necessary  or  what  func- 
tion, if  any,  they  might  have.  Experiments  were  therefore  devised 
to  test  the  relationship  of  these  molds  to  the  ripening  processes  out- 
lined above.  The  constant  occurrence  of  other  molds  upon  the  cheese 
brings  up  the  question,  How  and  to  what  extent  do  the  latter  affect 
the  ripening  process  ?  The  experiments,  therefore,  have  been  made  to 
include  as  many  species  as  possible.  Where  detailed  chemical  analy- 
ses had  to  be  made  the  work  has  necessarily  been  restricted  to  a  few 
forms. 

For  this  purpose,  in  addition  to  the  Camembert  Penicillium  and 
Oidium  lactis,  the  Penicillium  found  in  Roquefort  cheese  ("der  Edel- 
pilz"  of  German  authors)  has  been  generally  used.  For  convenience 
it  is  called  the  "  Roquefort  Penicillium  "  or  "  Roquefort  mold."  One  of 
the  Mucors,  probably  Mucor  or  Chlamydomucor  racemosus,  is  so  com- 
monly found  that  it  has  often  been  included.  A  pure  white  mold 
closely  related  to  the  Camembert  Penicillium  has  given  some  inter- 
esting contrasts.  When  reference  is  made  to  any  of  the  numerous 
undetermined  green  species  of  Penicillium,  they  will  be  indicated  by 
the  letter  or  number  under  which  they  appear  in  the  record  book  of 
cultures,  and  under  which  the  origin  and  subsequent  cultural  history 
of  all  species  studied  has  been  kept. 

OUTLINE    OF   THE    WORK. 

These  studies  involve  two  classes  of  data,  first,  those  experiments 
requiring  quantitative  analyses,  which  have  been  conducted  in 
cooperation  with  Mr.  A.  W.  Bosworth,  chemist  to  this  investigation, 
the  results  of  which  series  of  analyses  will  appear  in  his  report;  second, 
experiments  which  show  the  physiological  characters  of  the  fungi  by 
physical  changes  in  the  appearance,  texture,  or  color  of  the  medium 
used,  or  by  the  production  of  flavors. 

The  results  may  be  anticipated  here  by  noting  that  these  two  classes 
of  data  did  not  prove  mutually  interdependent,  but  that  analysis  may 
show  in  general  the  right  stage  of  chemical  changes  called  for  in  a  ripe 


12  FUNGI    IN    CHEESE    RIPENING. 

cheese  without  the  necessary  texture  and  flavor;  and,  conversely,  the 
practically  necessary  texture  and  flavor  may  be  obtained  in  a  cheese 
differing  considerably  in  its  chemical  characteristics  from  the  standard 
market  article.  In  our  practical  experiments  we  sought  first  for 
proper  appearance,  texture,  and  flavor  of  the  cheeses;  then,  without 
disturbing  these,  endeavored  so  to  control  the  processes  of  ripening  as 
to  satisfy  the  standard  of  chemical  composition  established  from  the 
study  of  market  cheeses. 

RELATION    OF   MOLDS   TO    ACIDITY. 

The  development  of  lactic  acid  has  been  shown  to  be  of  primary  im- 
portance in  the  control  of  deleterious  bacteria.  In  our  previous  paper 
it  has  also  been  seen  that  after  doing  its  work  this  acidity  gradually 
disappears  in  the  ripening  process.  The  disappearance  of  the  acid 
has  been  attributed  by  Roger,8  by  Epstein,8  and  by  Maze 9  to  the  activ- 
ity of  molds,  and  interpreted  as  preparing  the  way  for  the  action  of 
peptonizing  bacteria.  This  view  of  the  relation  of  molds  to  cheese 
ripening  has  been  widely  quoted  as  their  only  function  in  the  process. 

The  acid  exerts  practically  no  selective  action  upon  any  of  the  molds 
studied.  Stoll  has  recently  shown  that  species  of  Penicillium  grow 
readily  in  media  containing  a  much  higher  percentage  of  acid  than 
ever  occurs  in  cheese  work.  The  use  of  acid  in  fungous  cultures  to  re- 
strain bacteria  is  practically  universal,  but  the  action  of  the  different 
species  of  mold  upon  the  acid  is  very  different.  This  is  strikingly 
shown  by  the  introduction  of  a  solution  of  litmus  into  the  culture 
media  used.  Litmus  gelatin  or  litmus  agar  may  be  a  deep  blue  if  used 
at  15  acid  on  Fuller's  scale,  as  is  usual  for  bacterial  studies,  or  a  clear 
bright  red  if  2  to  4  drops  of  normal  lactic  or  other  acid  are  added  to 
10  c.  c.  of  medium.  No  mold  cultivated  in  this  work  has  failed  to 
show  some  definite  relation  to  acidity  indicated  by  litmus  reaction. 
Some  fungi,  as  soon  as  they  develop  visible  colonies,  begin  to  change 
red  (acid)  media  to  blue  (alkaline),  and  consistently  maintain  this 
character.  Many  others,  when  grown  in  blue  gelatin  (designating  by 
blue  gelatin  15  points  acid  to  phenolphthalein=10  points  alkaline  to 
litmus  on  Fuller's  scale),  begin  by  changing  the  blue  to  red.  This 
change  may  vary  from  the  faintest  tinge  of  red  in  only  that  part  of  the 
medium  directly  in  contact  with  the  threads  of  the  young  colony  to 
deep  red  over  large  areas.  Oidium  lactis  and  Roquefort  Penicillium 
produce  at  times  a  very  slight  pink,  which  barely  traces  the  outer  limits 
of  the  young  colonies  before  the  blue  reaction  begins  to  appear.  At 
other  times  the  red,  if  appearing  at  all,  has  been  so  evanescent  as  to  be 
overlooked.  It  has  been  suggested  that  this  slight  appearance  of 
acidity  might  be  due  to  the  excretion  of  carbon  dioxide  in  respiration, 
which,  although  continuous,  is  afterwards  masked  by  many  times 
larger  changes  in  other  substances. 


CAMEMBERT    AND    ROQUEFORT.  13 

The  Camembert  Penicillium,  and  several  of  the  very  common 
green  species  of  Penicillium,  when  grown  upon  blue  gelatin,  at  first 
turn  all  the  substratum  in  contact  with  the  growing  colonies  to  a 
bright  red.  Some  species  produce  areas  of  red  beyond  the  limits  of 
the  mycelium.  These  effects  are  most  clearly  seen  by  examining  the 
colony  from  the  under  side.  Later  a  spot  of  blue  appears  in  the  cen- 
ter of  the  colony  below  and  gradually  extends  outward  until  com- 
monly the  entire  mass  of  culture  medium  has  become  blue.  This 
often  involves  a  change  of  reaction  in  agar  or  gelatin  2  to  3  cm. 
beyond  the  colony.  It  is  thus  clear  that  there  must  be  either  the 
secretion  or  the  excretion  by  the  mycelium  into  the  medium  of  a 
substance  capable  of  changing  this  reaction  or  the  absorption  from 
the  medium  of  some  substance,  thus  changing  its  reaction.  The 
exact  nature  of  this  change  has  not  been  determined.  Increase  in  the 
percentage  of  acidity  or  of  alkalinity  retards  the  change  of  reaction. 
In  certain  experiments  phenolphthalein  was  introduced  into  red 
litmus  media  and  several  species  of  Penicillium  and  Oidium  lactis 
were  grown  upon  it.  With  the  Camembert  Penicillium  the  entire 
mass  of  agar  became  blue  in  a  few  days,  and  remained  so  for  nearly 
three  weeks.  Then  the  characteristic  pink  color  for  the  alkaline 
reaction  of  phenolphthalein  appeared  on  the  under  side  of  the 
colony.  This  was  tested  by  opening  the  colony  with  a  platinum 
needle  and  introducing  a  very  small  drop  of  normal  acid,  when  the 
pink  area  was  changed  first  to  blue  and  then  to  red.  As  the  acid 
diffused  outward  from  the  center  the  wave  of  blue  traveled  outward, 
being  replaced  constantly  by  red  until  all  trace  of  the  phenolphthalein 
reaction  was  gone.  The  other  species  used  did  not  give  this  reaction. 
There  are  forms  including  some  species  of  Penicillium,  Aspergillus 
niger,  Monilia  fructigena,  and  others,  which  produce  the  acid  reaction 
in  litmus  media  without  any  change  to  blue.  Several  species  of  Peni- 
cillium rapidly  produce  the  purplish  color  which  is  characteristic  of 
the  turning  point  of  litmus  at  which  their  further  development 
occurs.  Apparently  these  bring  acid  or  alkaline  media  to  that  point 
without  further  change.  It  would  appear,  then,  that  the  relations  of 
these  molds  to  acidity,  as  indicated  by  the  litmus  reaction,  is  reason- 
ably uniform.  To  determine  whether  the  litmus  reaction  would  be 
reliable  upon  a  medium  closely  allied  to  cheese,  test  tubes  of  sepa- 
rated milk  were  prepared,  blue  litmus  added,  and  the  tubes  sterilized. 
Eleven  species  of  Penicillium  were  inoculated  into  these  tubes  and 
observations  made  every  day.  Of  the  eleven  species,  four,  including 
the  Camembert  Penicillium,  produced  a  layer  of  red  milk  for  a  few 
millimeters  below  the  colonies,  which  later  was  changed  back  to  blue. 
The  other  species  either  intensified  the  blue  or  produced  no  change. 

The  suggestion  has  been  made,  that  neutralization  of  acid  is  due 
to  the  production  of  ammonia.     A  series  of  cultures  were  made  in 


14  FUNGI    IN    CHEESE    RIPENING. 

cooperation  with  Mr.  A.  W.  Bosworth  to  test  the  production  of  ammo- 
nia compounds  by  mold  action.  The  species  used  were  the  Roquefort 
Penicillium,  the  Camembert  Penicillium,  Penicillium  sp.  (record  No. 
310),  Oidium  lactis,  Oidiwn  sp.  (record  B),  and  Aspergillus  niger. 
These  were  grown  upon  potato  ager,  to  which  litmus  and  lactic  acid 
were  added.  The  Aspergillus  culture  remained  bright  red;  all  the 
others  became  deep  blue.  Upon  analysis  the  Aspergillus  niger  was 
found  to  have  produced  the  largest  amount  of  ammonia.  Study  of 
the  figures  showed  that  the  ammonia  alone  was  not  sufficient  to  neu- 
tralize the  acid  used  in  any  case.  It  is  clear,  then,  that  the  lactic  acid 
must  have  been  neutralized  by  some  other  basic  products  of  digestion 
rather  than  by  ammonia.  If  the  acid  were  absorbed  and  dissociated 
after  absorption  the  area  of  blue  would  be  restricted  to  the  neighbor- 
hood of  the  hyphas,  or  the  diffusion  of  the  acid  for  considerable  dis- 
tances would  produce  purple  tones  instead  of  sharply  marked  areas 
of  red  and  blue.  The  data  seem  to  indicate  that  chemical  decompo- 
sition or  neutralization  of  acid  must  be  the  action  of  some  product 
excreted  by  the  fungus,  probably  an  enzyme. 

It  has  thus  been  shown  by  many  experiments  that  the  Camembert 
Penicillium  and  Oidium  lactis  are  two  of  many  species  capable  of  reduc- 
ing the  acidity  of  the  media  upon  which  they  grow.  Many  other  species 
of  the  same  genus  produce  this  effect  more  quickly  than  the  Camembert 
Penicillium  and  some  act  at  about  the  same  rate.  The  reduction  of 
the  acidity  of  the  cheese  may  clearly  be  attributed  to  these  molds; 
but  the  study  of  the  relations  of  many  other  molds  to  acid  indicates 
that  any  of  a  large  number  of  species  might  be  equally  or  more  useful 
for  the  accomplishment  of  this  step  in  cheese  ripening.  If,  there- 
fore, these  particular  molds  are  essential  to  Camembert  cheese  ripen- 
ing, their  special  function  must  be  sought  in  other  steps  of  the 
process. 

THE    BREAKING    DOWN    OF    CASEIN. 

The  changes  in  firm  sour  curd  which  result  in  the  production  of  the 
soft,  buttery,  or  semiliquid  texture  of  the  Camembert  cheese  present 
some  very  complex  problems.  These  may  be  grouped  as  (1)  the 
purely  chemical  questions,  which  involve  qualitative  and  quantitative 
analyses  of  the  material  at  every  stage;  (2)  The  biological  and 
physical  questions,  which  deal  with  the  agents  and  conditions  which 
produce  these  results  and  with  the  gross  appearances  of  the  final 
products,  whose  descriptions  do  not  depend  upon  detailed  chemical 
analysis. 

(1)  The  chemist  describes  the  general  course  and  extent  of  these 
processes !  as  a  change  in  which  the  insoluble  or  but  slightly  soluble 
compounds  of  casein  found  in  sour  curd  are  rendered  almost  com- 
pletely soluble  in  water.  The  details  of  the  process  and  the  data  will 
appear  later  in  the  report  of  the  chemist. 


CAMEMBERT    AND    ROQUEFORT.  15 

(2)  To  determine  what  relation  the  molds  might  have  to  this  change 
involved  a  great  many  cultures  on  different  media.  In  some  experi- 
ments the  number  of  species  used  was  large  and  the  results  acquired 
in  that  way  a  comparative  value,  but  in  the  more  complicated  trials 
the  work  was  limited  to  those  mentioned  above. 

It  is  practically  impossible  to  produce  a  normal  cheese  in  such  a 
way  as  to  avoid  contamination  with  bacteria  or  molds.  It  is  difficult, 
therefore,  to  study  directly  upon  cheese  the  relations  of  organisms  to 
the  steps  of  cheese  ripening.  Even  were  this  possible,  the  complexity 
of  the  changes  encountered  would  make  the  interpretation  of  the  phe- 
nomena difficult.  The  activities  of  these  molds  have,  therefore,  been 
studied  in  pure  culture  upon  a  series  of  media  which  would  give  infor- 
mation as  to  steps  of  the  process.  While  these  cultural  studies  were 
proceeding,  many  cheeses  were  made  and  inoculated  with  the  Camem- 
bert  and  Roquefort  Penicillia.  The  measure  of  success  obtained  from 
cheese  inoculated  with  the  Camembert  Penicillium  gave  good,  practi- 
cal ground  for  its  continued  study.  These  detail  studies  may  be  dis- 
cussed best  separately. 

LIQUEFACTION    OF    GELATIN. 

The  liquefaction  of  gelatin  media  has  been  much  used  as  an  index  of 
digestive  activity.  All  species  obtained  have  been  grown  upon  neu- 
tral and  acid  sugar  gelatin  and  the  effects  noted  carefully. 

The  difference  in  action  between  the  molds  important  in  this  inves- 
tigation are  striking.  The  Mucor  produces  a  slow  but  rather  com- 
plete liquefaction;  Oidium  lactis  will  gradually  soften  the  gelatin  so 
that  the  center  of  the  colony  is  liquefied;  a  pigment-producing  Peni- 
cillium (recorded  simply  as  O)  will  liquefy  all  the  gelatin  in  contact 
with  it  so  quickly  that  it  becomes  in  a  week  a  floating  colony  in  a 
watery  pool  twice  its  own  diameter.  Several  other  species  of  Peni- 
cillium have  the  same  effect.  The  Roquefort  Penicillium  softens  gela- 
tin somewhat,  but  never  produces  a  watery  liquefaction.  The 
Camembert  Penicillium  often  produces  a  slight  liquefaction  under  the 
center  of  the  colony,  but  never  extends  that  liquid  area  to  half  the 
total  size  of  the  colony.  This  seems  to  indicate  that  the  Penicillium  O 
and  its  allies  would  produce  a  rapid  digestion,  that  the  Mucor  would 
be  somewhat  slower,  that  the  Camembert  mold  might  have  some  diges- 
tive effect  and  the  Roquefort  mold  very  little,  if  any,  value.  The  test 
of  the  ability  to  liquefy  the  gelatin  used  gives,  therefore,  only  indefi- 
nite or  negative  results  as  to  any  advantageous  relation  of  these  par- 
ticular species  to  cheeSe  ripening. 

Comparative  study  of  numerous  cultures  of  many  species  of  fungi 
upon  gelatin  gives,  however,  some  very  interesting  suggestions.  In 
many  species  which  liquefy  litmus  gelatin  rapidly,  the  area  of  liquefac- 
tion is  surrounded  by  a  blue  (alkaline)  band.     For  example,  in  one 


16  FUNGI    IN    CHEESE    RIPENING. 

experiment  with  Penicillium  392  at  its  most  active  period  of  growth  a 
colony  15  mm.  in  diameter  was  surrounded  by  a  liquefied  area  4  to  8 
mm.  wide.  This  area  was  in  turn  surrounded  by  a  band  of  intense 
blue  shading  gradually  in  a  width  of  perhaps  10  mm.  into  unchanged 
red  litmus  gelatin.  The  medium  which  had  been  liquefied  was  almosi 
colorless. 

Several  suggestions  may  be  drawn  from  many  such  observations. 
The  change  in  acidity  of  the  medium,  as  has  been  noted  above,  may 
be  effected  at  a  distance  of  2  to  3  cm.  from  the  colony.  This  change 
of  litmus  reaction  advances  faster  than  the  area  of  liquefaction  of 
the  gelatin.  The  breadth  of  the  area  of  liquefaction  shows  that  the 
action  of  the  fungus  is  not  a  digestion  by  contact,  but  the  secretion 
into  the  medium  of  diffusible  agents,  that  is,  enzymes.  In  most  of 
these  species  liquefaction  occurs  only  in  areas  having  alkaline  reac- 
tion. No  general  relation  between  acidity  and  digestion  is  estab- 
lished. The  substantial  uniformity  of  the  results  of  repeated  cultures 
of  the  same  species  of  fungi  upon  gelatin  made  after  the  formula  used 
established  its  usefulness  as  a  test  of  the  ability  of  an  organism  to  per- 
form this  particular  digestion.  It  will  be  shown  later  that  the  ability 
to  liquefy  this  variety  of  gelatin  is  not  to  be  regarded  as  a  general  test 
of  the  ability  of  a  species  to  produce  active  proteolytic  enzymes. 

raulin's  fluid. 

To  test  the  ability  of  these  species  to  grow  in  a  medium  entirely  lack- 
ing in  proteid,  Raulin's  fluid  was  used  as  given  by  Smith  and  Swingle,10 
but  modified  by  leaving  out  the  potassium  silicate  and  zinc  sulphate. 
Sterilized  flasks  of  this  solution  were  inoculated  with  Mucor,  Oidium 
lactis,  Camembert  Penicillium,  and  Roquefort  Penicillium.  All  four 
grew.  The  Oidium  lactis  and  Mucor  did  not  appear  to  develop  in  an 
entirely  normal  way.  Both  species  of  Penicillium  grew  richly  and 
fruited  normally.  The  culture  of  the  Camembert  mold,  after  growing 
several  weeks,  was  examined  chemically  and  digestive  experiments 
conducted  by  Mr.  Bosworth  demonstrated  the  presence  of  a  proteolytic 
enzyme.  In  this  way  it  was  shown  that  this  fungus  could  not  only 
construct  proteid  from  inorganic  compounds  of  nitrogen,  but  would 
produce  proteolytic  enzymes  in  such  a  solution.  Enzyme  studies 
were  not  made  for  the  other  species  used  in  this  experiment. 

CASEIN. 

For  a  medium  at  the  opposite  extreme,  the  chemists  prepared  pure 
casein.  This  was  weighed  into  2-gram  lots,  moistened,  sterilized  in 
the  autoclave,  and  inoculated  with  five  species  of  mold.  All  grew  and 
fruited  luxuriantly.  This  experiment  showed  only  that  the  species 
used  were  able  to  break  down  casein  and  to  grow  normally  upon  the 
products  of  this  digestion  without  the  addition  of  other  nutrients. 


CAMEMBERT    AND    ROQUEFORT.  17 

STERILE  MILK  AND   CURD. 

Sterilized  milk  and  sterilized  curd  offer  a  substratum  related  to 
cheese.  Sterilized  milk  in  quantities  varying  from  40  c.  c.  to  150  c.  c. 
in  test  tubes  and  Erlenmeyer  flasks  has  often  been  used.  Nearly  all 
species  of  Penicillium  grow  luxuriantly,  forming  a  felted  mass  of 
mycelium  often  2  to  4  mm.  in  thickness  upon  the  surface  of  the  milk. 
With  the  absorption  of  the  milk  in  such  cultures  of  the  Camembert 
and  Roquefort  species  the  mass  of  mycelium  buckles  and  bends, 
tubercles  of  mycelium  arise  on  the  under  side  of  the  mass  and  grow 
downward,  keeping  the  mold  in  connection  with  the  fluid.  In  this 
way  a  culture  may  continue  to  grow  for  several  months  until  it  forms 
tough,  irregular  masses  of  felted  hyphae,  filling  the  test  tube  for  an 
inch  or  more  downward  from  the  original  surface  of  the  milk.  The 
milk  below  the  colony  soon  becomes  transparent,  giving  reactions  for 
digestion,  with  a  residue  of  curd  at  the  bottom,  which  in  the  course  of 
time  may  be  almost  completely  dissolved.  With  the  Oidium  lactis, 
on  the  contrary,  the  colonies  largely  sink  below  the  surface,  so  that  the 
milk  may  be  quite  well  filled  with  mycelium  upon  which  chains  of 
spores  are  only  produced  in  quantity  at  or  just  below  the  surface. 
Similar  experiments  with  100  grams  of  sterilized  curd  in  flasks,  inocu- 
lated with  the  Camembert  and  Roquefort  molds,  have  shown  that 
either  species  is  able  to  change  the  chemical  composition  until  the 
derivatives  of  casein  are  amost  completely  water  soluble.  Such  cul- 
tures were  plated  to  show  their  freedom  from  contamination  by  bac- 
teria before  analysis.  The  resulting  products  give  the  standard  reac- 
tions for  digestion.  These  experiments  show  that  either  of  these 
molds  is  capable  of  producing  digestive  changes  comparable  in  their 
completeness,  rapidity,  and  general  nature  to  those  shown  by  analysis 
to  have  occurred  in  the  ripening  of  Camembert  cheeses. 

DOES  THE  MYCELIUM  PENETRATE  THE  CHEESE? 

It  must  be  noted  carefully  that  this  action  of  the  Camembert  mold 
goes  on  without  the  complete  penetration  of  the  substratum  by  the 
mycelium  of  the  mold.  That  this  is  true  is  readily  seen  in  milk  cul- 
tures, where  the  limits  of  the  development  of  the  mycelium  are  sharp 
and  clear.  The  same  fact  has  been  demonstrated  for  cheese  by  hun- 
dreds of  sections  and  careful  cultural  studies  many  times  repeated. 
The  mycelium  forms  a  dense  mat  upon  the  surface  of  the  fluid  or  the 
mass  of  curd,  or  the  newly  made  cheese.  It  follows  the  irregularities 
of  the  surface  and  is  not  found  to  enter  well-packed  curd  to  anj^  extent. 
It  is  very  difficult  to  prove  that  hyphae  of  this  mold  actually  appear  in 
curd  of  uniform  texture  below  1  or  2  mm.  When  found  deeper,  careful 
search  usually  shows  a  cracking  of  the  surface,  so  that  the  mycelium 
may  follow  the  opening  already  made.  In  no  case  of  many  hundreds 
21156— No.  82—06 3 


18  FUNGI    IN    CHEESE    RIPENING. 

of  cheeses  studied  and  experiments  performed  has  the  mold  been 
found  to  fruit  in  cavities  not  opening  broadly  upon  the  surface.  This 
is  in  marked  contrast  to  the  habit  of  the  Penicillium  instrumental  in 
the  ripening  of  Roquefort  cheese,  which  penetrates  the  channels  of  the 
substratum  and  fruits  in  every  cavity  large  enough  to  accommodate  a 
conidiophore.  The  Roquefort  mold  will  make  every  cavity  in  a 
cracker  or  piece  of  bread  green  with  spores,  while  the  Camembert 
mold  will  fruit  upon  the  surface  of  the  bread  or  cracker  with  only 
vegetative  mycelium  inside  the  bread. 

Definite  experiments  to  prove  that  this  digestive  power  on  the  part 
of  the  Penicillium  is  due  to  the  secretion  of  one  or  more  enzymes  have 
given  characteristic  reactions  for  digestion  many  times.  Without 
here  discussing  these,  chemical  reactions,  it  has  been  shown  that  the 
chemical  action  of  the  fungus  is  carried  on  at  distances  from  the 
mycelium  which  preclude  direct  action.  The  enzyme  must  therefore 
be  secreted  and  diffuse  outward  from  the  mycelium  into  the  sub- 
stratum. This  explains  why  the  Camembert  cheese  begins  to  ripen 
just  under  the  surface  and  the  process  progresses  inward  from  all 
sides  until  the  cheese  is  entirely  ripe.  Before  this  process  is  complete 
the  center  is  simply  sour  curd.  A  good  illustration  of  this  action  is 
seen  in  cheeses  which  are  ripened  without  turning.  In  such  cases  the 
development  of  mold  and  enzyme  on  the  lower  surface  is  prevented, 
and  as  a  consequence  ripening  is  delayed  on  that  surface. 

CAMEMBERT   PENICILLIUM   UPON    CHEESE. 

Many  cheeses  have  been  made  and  inoculated  with  this  mold  in  con- 
junction with  pure  cultures  of  lactic  starter.  Little  difficulty  is  found 
in  this,  since,  if  an  abundance  of  spores  are  put  upon  the  cheese  when 
made,  this  mold  seems  capable  of  taking  and  maintaining  the  lead  of 
all  others.  A  cheese  made  in  this  way  and  ripened  for  from  three  to 
four  weeks  will  finally  be  rendered  creamy,  or,  under  some  conditions, 
waxy^  throughout,  in  color  white  within,  in  flavor  almost  neutral, 
having  no  particular  character — good  or  bad — and  hence,  to  one  fond 
of  Camembert  cheese,  tasteless  and  insipid.  The  important  fea- 
tures of  this  ripening  process  are,  then,  the  completeness  of  its  action 
and  the  entire  absence  of  any  objectionable  character  in  its  flavor. 
Biological  analysis  has  shown  that  the  center  of  such  a  ripened  cheese 
may  be  practically  a  pure  culture  of  lactic  organisms.  The  texture  is, 
therefore,  obtainable  by  the  use  of  the  Penicillium  alone. 

COMPARATIVE    STUDIES   OF   FUNGOUS    DIGESTION. 

Comparative  tests  of  digestive  action  have  been  made  for  a  number 
of  molds.  The  Roquefort  Penicillium  has  been  used  in  parallel  cul- 
tures with  the  Camembert  Penicillium  in  many  determinations.     It 


CAMEMBERT    AND    ROQUEFORT. 


19 


has  shown  equal  or  greater  ability  to  digest  milk  and  curd.  A  typ- 
ical example  of  several  series  consisted  of  the  cultivation  of  1 1  species 
of  Penicillium  upon  sterilized  milk  in  large  test  tubes.  Observation 
of  results  after  seven  days  showed  digestion  by  7  of  these  species.  In 
5  of  them  the  amount  of  action  exceeded  that  of  the  Camembert  Peni- 
cillium, and  some  of  them  appeared  to  digest  milk  at  least  twice  as 
rapidly  as  did  that  species  in  the  first  week. 

In  another  series  milk  agar  was  made  by  dissolving  1  to  2  per  cent 
of  the  agar  in  water  at  130°  C.  and  pouring  together  equal  quantities  of 
the  hot  agar  and  hot  sterilized  milk.  If  poured  into  Petri  dishes  at 
once  this  medium  was  smooth  and  clear,  but  if  acidified  or  sterilized 
after  mixing,  flakes  of  precipitate  appeared.  The  flaky  precipitate  in 
the  acidified  cultures  was  found  very  useful  as  an  indication  of  diges- 
tion. In  cultures  upon  the  surface  of  such  plates  where  digestive 
action  was  strong  the  flakes  would  entirely  disappear.  Twenty- 
three  species  of  mold  were  tested  upon  milk  agar  in  this  way.  Of 
these,  8  produced  a  distinctly  stronger  digestion  than  the  Camembert 
Penicillium;  5  produced  digestion  approximately  equaling  that  spe- 
cies, and  10  produced  less  digestion.  These  cultures  were  mostly 
made  in  duplicate,  and  both  results  in  all  but  two  cases  agreed  fully. 
Oidium  lactis  produced  comparatively  little  effect  upon  this  medium. 

Table  I. — Reaction  of  certain  species  of  molds. 


Species. 


Litmus. 


132. 

310. 


Camembert  P Red,  then  blue. 

Roquefort  P Blue 


Oidium... 
Mucor  12.. 
Mucor  191 . 

O 

300 


Blue 

Blue 

Blue 

Blue 

Blue 

Red,   then  slowly 

blue. 
Red,  then  blue 

Red,  then  blue 

Blue 


Monilia  Candida 
19K. 

P.  brevicaule 

:v.y> 

240 

Aspergillus  niger.    Red 

13.") Blue 

136 !  Red  to  purple  blue. 


Blue. 
Blue. 
Blue. 


Liquefaction 
oi  gelatin. 


Partial . 


Softening 

Incomplete.. 
Incomplete.. 
Incomplete. . 

Rapid 

Partial 


Slight . . 

Slight.. 

Partial. 
Rapid . . 


Rapid 

Rapid 

Rapid 

Rapid 

Rapid 

Partial    soft- 
ening. 


Rate  of 
digestion 
ofcurd. 


Medium.. 
Rapid  . . . 


Rate  of  diges- 
tion of  milk. 


5°  to  10°  C. 


Medium. 
Rapid . . 
Slow.... 


Slow. 
Slow. 
Slow. 

Rapid Rapid . 

M  e  d  i  ti  m  j  Rapid . 
to  rapid. 
Medium 


Grow,  slow  fruit- 
ing. 

Characteristic 
growth. 

Characteristic. 

Poor  growth. 


Retarded. 


Slow  to  '  Slight Slow  growth. 

medium. 

Slow Medium Slow  fruiting. 

Rapid I  Rapid 


Rapid Rapid 

Slight Rather  slow. 

Rapid 

Slow 

Rapid 

Medium 


Characteristic. 


Two  species  of  Penicillia,  68  and  310,  found  closely  associated  upon 
cheese  with  the  Camembert  Penicillium,  produced  little  digestion. 
The  Roquefort  Penicillium  and  several  other  molds  often  found  upon 
Camembert  cheese  appeared  to  act  much  more  rapidly  than  the  Ca- 
membert mold  itself. 


20  FUNGI   IN    CHEESE    RIPENING. 

All  of  these  series  of  cultures  under  different  conditions  have  many 
times  shown  the  same  results  and  prove  that  the  ability  to  digest  curd 
is  common  to  many  species  of  fungi.  The  species  we  have  been  led  to 
call  the  Camembert  Penicillium  possesses  this  character  in  common 
with  numerous  other  molds,  many  of  which  act  more  rapidly  than 
this  one. 

After  the  ability  of  several  molds  to  digest  curd  is  established,  the 
relation  of  any  particular  mold  to  cheese  ripening  must  be  determined 
by  the  character  of  the  products  of  that  digestion  and  the  flavors  asso- 
ciated with  it.  No  pure  culture  upon  a  medium  previously  sterilized 
by  heat  has  given  a  taste  resembling  that  of  Camembert  cheese. 
Cheese  made  and  kept  in  an  atmosphere  of  chloroform,  which  pre- 
vented mold  and  bacterial  development,  refused  to  ripen.  Numerous 
cheeses  made  and  not  inoculated  with  molds  have  uniformly  failed  to 
develop  the  texture  and  flavor  of  Camembert  cheese,  although  such 
cheeses  have  usually  become  covered  with  molds  of  various  species. 
The  type  of  cheese  made  and  sold  in  this  country  as  Isigny  and  Brie, 
and  sometimes  labeled  Camembert,  which  always  shows  Oidium  lactis 
associated  with  bacteria,  differs  entirely  in  appearance,  texture,  odor, 
and  flavor  from  Camembert;  yet  Oidium  lactis  is  capable  of  neutraliz- 
ing the  acid  of  the  cheese  much  more  rapidly  than  the  Camembert 
Penicillium.  Nevertheless  the  center  of  such  a  cheese  remains  acid 
for  a  longer  time  than  is  required  to  ripen  a  Camembert  cheese,  while 
the  texture  of  Camembert  is  not  produced.  The  necessity  for  the  pres- 
ence of  another  agent  in  this  ripening  is  clearly  established. 

More  than  2,000  cheeses  have  been  made  and  ripened  at  this  station 
with  the  Camembert  mold  under  varying  conditions.  Hundreds  of 
these  cheeses  have  shown  repeatedly  that  cheese  so  made  will  assume 
in  ripening  the  texture  of  the  best  imported  article.  The  Camembert 
Penicillium,  therefore,  is  seen  to  be  able  to  neutralize  the  acid  of  the 
freshly  made  cheese  and  to  produce  the  texture  desired,  but  not  the 
flavor.  It  remains  to  determine  whether  other  molds  may  not  be 
equally  useful  in  this  process.  For  comparison  cheeses  have  been 
made  and  inoculated  with  the  Roquefort  Penicillium  with  undeter- 
mined species  of  Penicillium  appearing  on  the  record  as  O,  300,  310,  68, 
132.  Of  these  species  one,  310,  when  cultivated  upon  every  medium 
used  except  the  cheese  duplicated  the  reactions  of  the  Camembert 
mold  completely.  Its  morphology  is  scarcely  distinguishable.  It 
differs  only  in  that  it  remains  pure  white  during  its  entire  cycle  of  de- 
velopment, while  the  Camembert  species  turns  gray-green  in  age.  The 
close  relationship  apparent,  together  with  a  promising  test,  led  to  its 
use  upon  over  100  cheeses.  The  breaking  down  resulting  from  its 
action  was  widely  different.  These  cheeses  were  drier,  waxy,  with  a 
mealy  crumbling  layer  just  under  the  rind.  The  physical  character 
of  the  results  and  the  flavor  produced  were  so  different  that  the 


CAMEMBERT    AND    ROQUEFORT.  21 

cheeses  were  entirely  worthless.  This  mold  was  originally  isolated 
from  a  market  Camembert  cheese,  where  it  was  found  mixed  with 
others. 

The  presence  of  the  Roquefort  Penicillium  may  be  seen  by  the  spots 
of  green  it  produces  and  may  be  detected  by  a  sharp,  bitter,  perhaps 
astringent,  taste.  The  texture  of  the  cheese  produced  is  different,  and 
the  flavor  when  it  is  present  in  any  large  amount  is  so  strong  as  to  be 
very  objectionable  to  many.  When  present  in  small  amounts  upon  a 
cheese  it  gives  a  certain  sharpness  or  piquancy  to  it,  such  as  has  been 
found  often  in  certain  brands  of  imported  cheese,  and  is  sought  for  by 
some  buyers. 

The  species  marked  O  and  300  secrete  a  bright  yellow  pigment  into 
the  cheese,  which  colors  every  area  with  which  it  comes  in  contact.  A 
cheese  was  inoculated  with  No.  300  and  examined  when  8  weeks  old. 
It  had  produced  no  trace  of  the  texture  of  Camembert.  The  center  of 
the  cheese  remained  practically  sour  curd,  while  the  portion  for  per- 
haps one-fourth  of  an  inch  under  the  colony  was  decomposed. 

The  species  marked  68  has  been  obtained  from  cheese  from  widely 
different  sources.  In  cultures  upon  milk  and  milk  agar  it  produced 
little  change.  A  cheese  inoculated  with  it  remained  largely  sour  curd 
for  two  months.  The  species  marked  132  is  a  very  common  green 
form,  appearing  in  dairy  and  other  cultures.  It  has  given  no  satisfac- 
tory results  when  grown  upon  cheese.  In  this  way  related  species 
found  in  cheese  work  have  been  tested  in  their  effects  upon  cheese  and 
shown  not  to  produce  digestion  comparable  in  physical  character  to 
that  demanded  in  a  Camembert  cheese  and  constantly  obtained  by 
the  use  of  the  Camembert  Penicillium.  There  seems  to  be  no  further 
question  that  this  species  of  Penicillium,  among  all  the  molds  so  far 
studied,  is  the  only  agent  capable  of  producing  the  characteristic 
texture  of  thp  best  type  of  Camembert  cheese,  with  no  objectionable 
flavors  or  colors. 

FLAVORS. 

All  attempts  to  produce  the  flavor  of  Camembert  cheese  in  pure  cul- 
tures upon  milk  and  curd  with  particular  organisms  have  failed.  Here 
again  we  have  had  to  depend  upon  the  use  of  cheeses  so  that  direct,  posi- 
tive proofs  have  not  been  possible.  The  value  of  the  indirect  or  cir- 
cumstantial evidence  offered  must  depend  upon  the  completeness  with 
which  all  factors  have  been  considered.  It  has  been  previously 
shown  that  a  cheese  may  be  ripened  to  the  texture  of  the  best  Camem- 
bert by  the  action  of  lactic  bacteria  and  the  Camembert  Penicillium, 
but  that  it  will  lack  flavor.  A  series  of  difficulties  are  met  here. 
The  typical  flavor  does  not  begin  to  appear  until  ripening  is  well 
along.  This  would  indicate  that  the  flavor-producing  agent  or  agents 
must  act  upon  already  partially  ripened  cheese  to  produce  the  par- 


22  FUNGI    IN    CHEESE    RIPENING. 

ticular  end  products  which  give  this  flavor.  But  coincident  with  this 
change  the  acidity  of  the  curd  has  become  so  far  reduced  that  bac- 
terial development  may  now  occur  on  the  surface  at  least,  and  as  a 
matter  of  observation  few  cheeses  begin  to  show  flavor  until  cultures 
from  their  surface  show  swarms  of  bacteria  of  various  species.  It  has 
not  been  practically  possible  to  change  these  conditions  sufficiently  to 
make  cheeses  bearing  only  pure  cultures  upon  the  surface.  The  prob- 
lem becomes,  then,  one  of  comparative  study  and  the  elimination  of 
the  unnecessary  factors  one  by  one,  rather  than  the  direct  produc- 
tion of  the  flavor  sought  in  a  single  conclusive  experiment. 

Some  organism  or  organisms  must  be  sought  for  to  produce  the 
flavor.  The  appearance  of  the  flavor  of  the  imported  article  in  cer- 
tain experimental  cheeses  at  this  stage  of  the  investigation  led  to 
their  immediate  study.  This  showed  that  Oidium  lactis  was  abundant 
upon  these  cheeses  and  emphasized  the  fact  that  it  had  always  ap- 
peared in  cultures  from  market  cheeses.  Oidium  had  been  excluded 
from  many  experiments  in  cheese  making  because  it  had  been  found  to 
be  associated  with  odors  that  seemed  undesirable,  as  well  as  because  of 
the  conclusion  of  Epstein  from  his  researches,  that  the  presence  of 
Oidium  is  uniformly  deleterious.  The  inoculation  with  spores  of 
Oidium  of  a  half-ripened  cheese  entirely  lacking  flavor  produced  the 
flavor  distinctly  in  a  single  week,  but  since  bacterial  action  seemed 
always  associated  with  this,  further  evidence  was  necessary.  Roger 
and  Epstein  have  attributed  the  ripening  of  Camembert  to  the  action 
of  certain  bacteria  without  distinguishing  that  the  production  of  the 
texture  of  the  cheese  is  accomplished  by  a  different  agent  from  the 
production  of  flavor.  In  their  descriptions  ripened  Camembert  is 
always  referred  to  as  slightly  reddish  in  color,  and  the  appearance  of 
this  color  is  regarded  as  an  indication  of  the  progress  of  ripening.  In 
cheeses  selected  and  forwarded  by  M.  Roger  this  red  color  was  very 
prominent  and  the  red  layer  was  found  to  consist  of  myriads  of  bac- 
teria of  a  few  species.  Cultures  from  these  cheeses  showed  that 
Oidium  lactis  was  also  present  in  abundance.  Numerous  tests  have 
been  made  with  the  bacteria  found  associated  with  the  various 
brands  of  Camembert  cheese  hitherto  without  producing  the  flavor  in 
any  case  independently  of  the  molds.  The  comparative  study  of 
many  cheeses  from  the  market  and  from  our  own  cellars  seems  to 
show  that  cheeses  may  have  the  typical  Camembert  flavor  without 
the  development  of  any  specific  surface  growth  of  bacteria.  The 
character  of  the  bacterial  growth  upon  the  surface  appears,  therefore, 
to  be  incidental  or  accidental,  though  its  presence  may  be  necessary 
to  exclude  air,  as  maintained  by  Maze9  in  a  recent  paper. 

Cheeses  of  good  flavor  have  been  produced  here  and  also  purchased 
in  the  market,  which  indicate  that  particular  surface  appearances  are 
not  essential  to  the  typical  flavor.     Similarly  the  introduction  into 


CAMEMBERT    AND    ROQUEFORT.  23 

new  cheeses  of  species  of  bacteria  found  in  cultures  from  the  interior 
of  good  cheeses  has  produced  either  no  effect  whatever  or  disagreeable 
flavors.  Thus  far,  therefore,  no  species  of  bacterium  has  been  found 
capable  of  producing  the  Camembert  flavor.  Although  the  flavor 
question  is  manifestly  still  unsettled,  we  may  offer  the  following  sum- 
mary of  the  data  at  hand  upon  relation  of  molds  to  flavor  in  Camem- 
bert cheese : 

(1)  Oidium  lactis  has  been  found  in  every  brand  of  Camembert 
cheese  studied. 

(2)  It  has  never  been  found  upon  a  ripened  Camembert  cheese  which 
lacked  the  flavor. 

(3)  The  flavor  has  never  been  found  in  a  cheese  without  the  Oidium. 

(4)  Every  other  species  with  which  the  flavor  seemed  obtainable  has 
been  eliminated  from  one  or  more  experiments  without  loss  of  flavor. 

(5)  Bacteria  or  other  molds  do  in  many  cases  modify  the  flavor  of 
Camembert  cheese,  but  do  not  seem  to  be  able  to  produce  it  inde- 
pendently of  the  mold.  There  thus  arise  characteristic  secondary 
flavors  which  are  associated  with  the  output  of  certain  factories  and 
which  command  special  markets.  These  varieties  are  usually  more 
highly  flavored  than  what  we  have  regarded  as  typical. 

The  essential  relation  of  the  Camembert  Penicillium  and  Oidium 
lactis  to  the  production  of  Camembert  cheese  is,  therefore,  well  estab- 
lished. Several  mycological  questions  remain:  What  are  the  opti- 
mum conditions  of  temperature  and  moisture  for  the  use  of  these 
molds  in  cheese  ripening?  What  are  the  most  practicable  means  of 
cultivating  material  for  inoculation?  How  can  the  proper  inocula- 
tion with  these  molds  be  most  effectually  secured  ?  What  other  fungi 
occur  as  contaminating  species  and  how  can  they  be  controlled? 

TEMPERATURE. 

Since  the  higher  temperatures  of  the  ripening  cellar  lead  more  rap- 
idly to  the  development  of  bacteria,  it  is  necessary  to  determine  the 
lowest  temperature  which  will  permit  mold  growth  and  also  enzyme 
action.  The  different  species  respond  quite  differently  to  tempera- 
ture. In  one  experiment  eight  species  were  inoculated  into  slanted 
tubes  of  gelatin  and  put  in  a  refrigerator  where  the  temperature 
varied  from  5°  to  10°  C.  Of  these  the  Camembert  Penicillium  and 
two  nearly  related  species,  Nos.  68  and  310,  grew,  but  fruited  very 
slowly,  showing  an  inhibiting  effect.  The  Roquefort  Penicillium 
grew  and  fruited  normally,  as  also  did  Oidium  lactis.  The  species  of 
Mucor  used  developed  very  slowly  and  fruited  only  slightly.  Two  of 
the  very  common  green  species  of  Penicillium  grew  richly.  Oidium 
lactis  grows  abundantly  in  the  Brie  and  Isigny  cellars  visited.  In 
those  the  temperature  was  50°  to  55°  F.  (11°  to  12°  C).  Numerous 
experiments  in  the  ripening  cellar  show  that  the  Camembert  Penicil- 


24  FUNGI    IN    CHEESE    RIPENING. 

lium  does  not  grow  its  best  in  a  room  cooler  than  60Q  F.  (15°  C),  and 
that  to  obtain  rapid  development  the  room  should  be  slightly  warmer. 
Until  this  mold  is  well  established,  therefore,  it  is  distinctly  an  advan- 
tage to  grow  it  at  a  temperature  of  65°  to  70°  F.  Repeated  experi- 
ments have  shown  that  lowering  the  temperature  to  52°  to  55°  F. 
checks  the  rate  of  ripening  very  materially.  A  difference  of  less  than 
10  degrees  between  two  rooms  will  often  make  as  much  as  two  weeks' 
difference  in  the  ripening  period  of  cheeses  from  the  same  lot  in  the 
two  rooms.  A  temperature  as  low  as  54°  to  55°  F.,  as  given  in  an 
article  in  the  Creamery  Journal  previously  referred  to,  appears  to  pro- 
long the  ripening  period  without  contributing  any  compensating 
advantages.  A  half-ripened  cheese  was  cut,  the  progress  of  the 
softening  of  the  curd  was  noted,  and  the  cheese  put  in  a  refrigerator, 
where  it  was  held  for  four  weeks  at  48°  F.  It  was  then  found  to  be 
completely  ripened  and  perhaps  a  little  old  in  one  place,  but  the 
changes  noted  at  the  end  of  this  period  would  have  been  produced 
within  a  single  week  at  60°  F.  The  cold-storage  possibilities  sug- 
gested by  this  experiment  will  be  further  studied. 

Some  experiments  were  made  to  show  the  resistance  of  spores  to 
heat.  The  spores  of  the  Camembert  and  Roquefort  Penicillia  were 
inoculated  into  gelatin  and  placed  in  an  incubator.  Heating  for  an 
hour  and  fifteen  minutes  at  56°  C.  killed  all  spores  of  the  Camembert 
species.  Only  a  few  spores  of  this  mold  grew  after  one  hour  at  the 
same  temperature,  while  some  spores  of  the  Roquefort  Penicillium 
grew  after  two  and  one-half  hours. 

HUMIDITY. 

The  use  of  very  moist  cellars  and  caves  in  the  ripening  of  this  class 
of  cheeses  is  practically  universal.  The  richest  development  of  mold 
is  seen  in  rooms  where  the  atmosphere  is  saturated  or  nearly  so.  This 
appears  to  be  exceptionally  true  for  species  like  the  Camembert  Peni- 
cillium, which  is  peculiarly  a  milk  fungus,  and  in  which  there  is  a 
large  development  of  thin-walled  aerial  mycelium.  So  dependent  is 
the  Camembert  mold  upon  abundance  of  moisture  that  it  has  been 
found  difficult  to  secure  a  rich  growth  upon  the  surface  of  a  cheese 
which  has  been  drained  for  two  or  three  days  before  inoculation.  Con- 
trary to  directions  commonly  given  for  ripening  these  cheeses,  which 
call  for  a  particular  degree  of  humidity,  cheeses  have  been  ripened 
successfully  in  our  cellars  at  the  saturation  point,  as  well  as  at  various 
degrees  of  humidity  below  that.  A  good  illustration  of  a  mold  which 
has  adapted  itself  to  changes  of  moisture  is  found  in  mold  No.  198 
Upon  a  fresh  cheese  in  a  moist  room  this  mold  forms  a  circular, 
ringlike  colony  of  floccose  hyphae  standing  often  8  mm.  high  upon 
the  surface  of  the  cheese.  In  a  drier  situation,  or  when  the  cheese 
is  nearly  ripe  and  the  rind  becomes  harder  and  dried,  the  same  mold 


CAMEMBERT    AND    ROQUEFORT.  25 

produces  conidiophores  which  barely  rise  above  the  substratum,  so 
that  the  surface  of  the  cheese  is  covered  by  a  white,  powdery  layer 
which  is  practically  pure  spores.  The  Mucors  are  so  sensitive  to  mois- 
ture that  they  scarcely  develop  upon  the  cheese,  except  sometimes 
during  the  first  few  days,  when  the  surfaces  are  very  wet.  They 
appear  to  be  unable  to  withstand  the  rate  at  which  surface  evapora- 
tion proceeds  in  the  ripening  cellars. 

INOCULATING    MATERIAL. 

The  problem  of  propagation  of  the  Camembert  Penicillium  for  inocu- 
lation purposes  presents  some  difficulties.  This  species  bears  spores 
only  upon  the  surface  of  the  culture  medium  used,  in  contrast  to  the 
Roquefort  species,  which,  when  grown  upon  bread,  develops  spores  in 
every  air  space,  as  well  as  on  the  surface.  To  produce  spores  in  quan- 
tity, therefore,  material  must  be  capable  of  sterilization  and  must  pre- 
sent the  largest  possible  amount  of  free  surface  in  proportion  to  the 
space  occupied.  For  the  preparation  of  such  material,  quart  fruit  jars 
have  been  used.  Various  styles  of  crackers  have  been  tried.  Most  of 
these  were  not  successful.  The  most  suitable  appears  to  be  the  hard, 
dry  "  water  cracker."  The  jar  is  filled  with  crackers  and  dry  sterilized 
at  140°  to  160°  C.  for  an  hour  or  more,  better  twice  on  successive  days. 
The  spores  may  be  added  directly,  or  first  inoculated  into  about  100 
c.  c.  of  sterile  water  (acidified  with  1.5  per  cent  of  lactic  acid  usually) 
and  this  poured  into  the  jar  and  shaken  until  all  the  crackers  are  wet. 
Various  types  of  "milk  cracker"  soften  to  a  pasty  mass  in  this  mois- 
tening process.  The  best  water  crackers  are  not  very  satisfactory, 
because  the  mycelium  tends  to  transform  bread  or  cracker  into  a  soft, 
gummy  mass.  The  crackers  become  matted  together  until  they  pre- 
sent much  less  actual  surface  than  might  be  expected.  The  substi- 
tutes tried  have  been  excelsior,  hay,  and  sheets  of  cardboard  wetted 
with  milk  or  whey.  Although  some  of  these  have  advantages,  they 
were  on  the  whole  less  satisfactory  than  the  water  crackers.  So  far, 
therefore,  on  account  of  the  very  different  habit  of  our  mold,  no  mate- 
rial has  been  found  so  easily  prepared  and  so  satisfactory  as  the 
"  Schimmelbrot "  of  the  Roquefort  cheese  makers. 

From  the  point  of  view  of  the  use  of  pure  cultures  the  Oidium  lactis 
is  even  more  troublesome.  This  mold  produces  a  large  proportion, 
and  in  some  strains  all  of  its  spores  as  chains  below  the  surface  of  the 
substratum.  For  pure-culture  work  Petri-dish  cultures  have  been  the 
only  satisfactory  vessels  used.  Its  exceedingly  rapid  development, 
however,  makes  possible  the  propagation  of  a  culture  from  day  to  day 
from  the  draining  boards  upon  which  the  cheese  is  made.  These 
become  heavily  coated  with  a  slimy  mass  of  mycelium  and  spores  upon 
standing  overnight.  Direct  transfers  from  them  have  been  used  with 
apparently  no  serious  trouble  from  contamination.     In  fact,  so  capa- 


26  FUNGI    IN    CHEESE    RIPENING. 

ble  is  the  Oidium  of  self-propagation  in  dairy  work  that  Epstein 
declares  it  to  be  present  in  all  dairy  work.  Although  Roger  in  his 
published  statement  does  not  mention  it  at  all,  it  was  found  abun- 
dantly upon  the  cheese  forwarded  by  him  to  this  station.  We  have 
succeeded  by  careful  work  in  making  many  cheeses  entirely  free  from 
Oidium,  but  with  the  ordinary  treatment  of  dairy  utensils  it  appears 
constantly  in  factory  practice.  It  is  practically  possible  to  rely  to  a 
considerable  extent  upon  the  ability  of  the  Oidium  to  propagate  itself, 
as  has  hitherto  been  done  in  the  factories. 

INOCULATION    WITH    PENICILLIUM. 

With  the  Penicillium,  however,  numerous  experiments  indicate  that 
there  is  much  advantage  in  early  and  effective  inoculation  from  cul- 
tures of  known  purity.  Whether  such  inoculation  must  be  always 
made  from  specially  grown  laboratory  cultures  is  questionable.  In 
factory  practice,  the  making  room  and  the  ripening  cellar  are  usually 
adjacent.  If  precautions  are  taken  always  to  have  on  hand  some 
cheeses  bearing  pure  cultures  (and  the  cheese  maker  must  know  his 
mold  so  well  that  there  will  be  no  question  about  it),  one  or  two  such 
cheeses  will  furnish  enough  inoculation  material  for  much  newly  made 
product.  This  would  be  indicated  by  the  rough  calculation  that  from 
the  abundance  of  the  chains  of  fruit  and  the  size  of  the  spores  (0.005 
mm.  in  diameter)  probably  about  enough  spores  are  produced  to 
cover  evenly  the  surface  upon  which  they  grow — perhaps  25,000,000 
to  the  square  inch.  Very  successful  inoculation  in  75  pounds  of  milk 
has  commonly  been  secured  by  tapping  a  Petri-dish  culture  over  the 
vat,  or  by  bifeaking  a  piece  of  cracker  about  an  inch  square  or  less  and 
stirring  it  into  the  milk. 

The  most  economical  and  successful  method  of  inoculation  so  far 
devised  has  been  the  use  of  a  sprinkling  jar  or  can.  For  this  purpose 
holes  1  mm.  or  less  in  diameter  in  the  jar  lid  are  demanded.  A  small 
amount  of  water  is  put  into  the  jar,  a  piece  of  cracker  or  cheese  covered 
with  mold  is  broken  into  the  water,  the  top  is  then  screwed  on,  and 
the  jar  thoroughly  shaken.  The  water  is  then  sprinkled  upon  the 
newly  made  cheese  at  the  time  of  first  turning,  so  that  both  sides  of 
each  cheese  receive  a  few  drops  of  water.  Excellent  results  have 
been  obtained  in  this  way  with  the  smallest  amount  of  inoculating 
material  and  the  least  requirement  of  labor  and  skill.  Such  a  jar 
should  be  emptied  and  washed  immediately  after  using.  The  mix- 
ture is  made  fresh  each  time.  Milk  may  be  used  instead  of  water,  as 
was  first  suggested  and  tried  by  Doctor  Conn;  but  the  water  has  been 
found  the  more  easily  managed.  The  practical  method  for  factory 
use  will  probably  vary  with  the  conditions  and  skill  of  the  maker. 


CAMEMBERT    AND    ROQUEFORT.  27 

VITALITY    OF    SPORES. 

Studies  have  been  made  upon  the  vitality  of  the  spores  of  the  spe- 
cies used.  This  varies  greatly  in  different  species.  In  some  of  the 
most  common  forms  spores  have  been  reputed  to  remain  viable  for 
several  years.  Recent  studies  by  Wehmer  showed  that  five  species 
of  Penicillium  used  in  his  experiment  were  entirely  dead  in  labora- 
tory cultures  at  the  end  of  two  and  one-half  years.  Cultures  of  the 
Camembert  Penicillium  grown  upon  potato  in  test  tubes  plugged  with 
cotton  have  refused  entirely  to  germinate  at  the  age  of  one  year. 
Other  -cultures  have  seemed  entirely  dead  inside  of  six  months.  In 
fact,  the  spores  of  this  mold  are  very  thin  walled  and  die  very  rapidly 
when  stored.  Under  such  conditions  they  lose  turbidity  and  become 
crenulated  or  indented.  Spores  of  Monilia  Candida  and  several 
others  have  grown  after  more  than  a  year  in  laboratory  cultures,  but 
their  germination  was  much  retarded.  Oidium  lactis  seems  to  be 
very  easily  killed  by  drying,  as  would  be  expected  from  a  species  with 
such  thin-walled  spores.  The  Roquefort  Penicillium  under  some  con- 
ditions is  more  resistant,  but  loses  vitality  quite  rapidly.  It  is  cer- 
tain, therefore,  that  to  give  the  best  results  material  for  inoculation 
should  be  fresh  and  vigorous.  Under  ordinary  circumstances  it 
would  not  be  desirable  to  use  material  more  than  a  few  weeks  old. 

CONTAMINATIONS. 

The  number  of  molds  found  upon  market  Camembert  cheese  shows 
the  need  of  care  in  guarding  against  contamination  of  cultures.  Ex- 
traneous molds  may  come  from  the  milk  or  from  the  utensils  used  or 
from  the  clothes  and  hands  of  the  workmen.  Although  the  milk^s 
the  primary  source  of  most  infections,  practical  experiments  haw 
shown  that  if  the  proper  molds  are  put  upon  the  cheese  at  the  time  of 
making  the  troubles  arising  in  this  way  may  be  minimized.  In  fact, 
sufficient  contamination -from  this  source  directly  to  ruin  a  cheese  is 
very  uncommon. 

The  very  habit  in  some  countries  of  washing  or  rinsing  cheese- 
making  utensils  in  whey  will  account  readily  for  the  universal  presence 
of  Oidium  lactis  and  perhaps  for  many  of  the  bacterial  infections  that 
result  in  loss.  But  the  source  of  the  most  trouble  in  a  cheese  cellar 
is  found  to  be  the  cheese  maker  himself.  The  cheeses  are  commonly 
exposed  upon  curing  boards  and  turned  and  examined  in  the  hands. 
In  this  way  spores  from  molds  or  bacteria  occurring  accidentally  as 
single  colonics  upon  single  cheeses  are  distributed  by  thousands  to 
hundreds  of  cheeses.  The  product  of  a  factory  may  almost  be  identi- 
fied in  the  markets  by  the  contaminations  upon  the  surface  of  its 
cheeses.  Certain  brands  of  the  cheese  always  bear  Monilia  Candida 
and  commonly  one  or  two  other  Monilias.  A  species  of  Fusarium  is 
distinctive  of  another  brand,  with  Acrostalagmus  cinnabarinus  occa- 


28  FUNGI    IN    CHEESE    RIPENING. 

sionally  present.  After  numerous  experiences  with  all  sorts  of  con- 
tamination this  trouble  has  been  practically  eliminated  from  our 
experimental  work  by  putting  the  fresh  cheeses,  as  soon  as  they  are 
drained,  salted,  and  comparatively  dry  upon  the  surface,  into  boxes 
which  are  slightly  larger  than  the  cheeses,  leaving  air  space  and  room 
for  mold  to  develop  normally.  In  this  way  fingering  is  done  away 
with,  the  cheese  is  turned  by  turning  the  box,  and  examined  by 
removing  the  lid  without  touching  the  surface,  so  that  a  colony  of 
mold  appearing  upon  one  cheese  is  no  longer  distributed  throughout 
the  cellar. 

It  is  therefore  possible  to  produce  cheeses  practically  free  from 
molds  other  than  those  inoculated  upon  their  surface.  Although 
such  boxing  upon  a  large  scale  may  be  practically  undesirable  on 
account  of  expense,  it  remains  certain  that  it  may  be  useful  in  elimi- 
nating certain  troubles  without  so  large  a  loss  as  would  come  from  dis- 
carding all  infected  cheeses,  many  of  which  would  ripen  satisfactorily 
but  for  the  danger  of  spreading  obnoxious  fungi  over  great  numbers  of 
cheeses. 

ROQUEFORT  CHEESE. 

The  well-known  Roquefort  cheese  is  another  highly  flavored  cheese 
in  which  mold  has  long  been  known  to  play  a  part.  In  manufacture 
this  cheese  approaches  the  hard  type,  but  the  ripened  cheese  bears  a 
closer  relation  to  the  softv cheeses.  Many  complete  descriptions  give 
the  details  of  its  making  and  curing.  These  need  not  be  repeated 
here.  Roquefort  is  by  description  a  goat's  or  sheep's  milk  cheese, 
made  in  France  principally,  though  cheese  of  nearly  the  same  quality 
is  said  to  be  made  in  other  parts  of  Europe  from  mixed  cow's  and 
sheep's  milk  or  from  cow's  milk  alone. 

The  great  popularity  of  Roquefort  cheese  makes  information  as  to 
the  biology  of  its  ripening  processes  very  desirable.  To  this  end  nu- 
merous specimens  of  Roquefort  have  been  purchased  and  analyzed. 
The  results  of  this  work  have  been  very  much  simpler  than  the  stud- 
ies of  Camembert.  The  ordinary  Roquefort  cheese  before  it  is  sent  to 
the  market  is  carefully  cleaned  and  covered  with  tin  foil.  Its  surface 
would,  therefore,  tell  very  little.  When  cut  it  is  seen  to  be  traversed 
by  channels  or  ho  es  made  by  the  prickle  machine  ( Stechmaschine) 
and  by  cracks.  Every  air  space  is  lined  with  green  Penicillium,  so 
that  the  cut  surface  is  said  to  be  marbled  with  green.  The  texture  of 
the  cheese  is  reasonably  uniform,  with  every  indication  that  ripening 
is  simultaneous  throughout  the  cheese  or  at  least  approximately  so. 
Its  texture  is  rather  crumbling  than  waxy,  with  a  tendency  to  dissolve 
readily  in  the  mouth.  The  taste  is  a  characteristic  sharp  flavor,  in 
which  a  rather  high  salt  content  is  noticeable.  Its  odor  is  strong, 
cheesy   rather   than    offensive   in    any  way,  except  as  pronounced 


CAMEMBERT    AND    ROQUEFORT.  29 

putrefactive  odors  are  sometimes  developed  in  the  rind.  Cultures  from 
the  surface  often  show  various  species  of  fungi.  There  is  no  regu- 
larity about  the  surface,  however,  while  uniformity  of  texture  and  ap- 
pearance is  universal  on  the  inside.  Cultures  from  the  interior  show 
a  remarkable  uniformity.  In  many  cheeses  examined  a  pure  culture 
of  a  single  species  of  Penicillium  has  been  found.  The  extremely  rare 
appearance  of  any  other  mold  in  the  cultures  has  been  remarkab  e. 
Similarly  the  bacterial  content  is  usually  limited  to  typical  lactic  forms. 
Sufficient  analyses  have  been  made  to  establish  clearly  that  a  first- 
class  Roquefort  cheese  should  conta  n  only  lact'c  bacteria  and  the 
Roquefort  Penicillium.  This  Penicillium  is  often  referred  to  by  writ- 
ers as  P.  glaucum  and  regarded  as  the  common  green  species,  but  as  it 
has  very  characteristic  morphological  and  physiological  characters  it 
seems  best  to  designate  it  as  the  Roquefort  Penicillium,  even  though 
it  quite  often  occurs  upon  other  substrata. 

The  cultures  which  have  been  conducted  in  connection  with  the 
study  of  Camembert  cheese  have  shown  that  the  Roquefort  Peni- 
cillium is  capable  of  digesting  curd  very  completely.  Here,  as  in 
Camembert  cheese,  chemical  analyses  have  shown  that  the  derivatives 
of  casein  become  almost  completely  water  soluble.  Further  pure- 
culture  experiments  upon  sterile  curd  have  shown  that  this  mold  dur- 
ing the  process  of  digestion  produces  bitter  flavors  during  the  first  few 
weeks,  but  that  its  continued  action  changes  these  to  typical  flavors 
of  the  Roquefort  cheese.  Here,  then,  we  have  a  definite,  positive  re- 
sult. It  is  thus  shown  that  the  Roquefort  Penicillium,  acting  with  the 
lactic  bacteria,  is  capable  of  ripening  Roquefort  cheese  without  the  in- 
troduction of  other  enzyme-producing  or  flavor-producing  organisms. 
The  investigations  of  the  chemical  nature  of  these  changes  have  barely 
been  touched  upon  at  this  time.  In  a  recent  experiment  a  cheese  of 
the  Roquefort  type  was  made  of  cow's  milk  inoculated  with  the 
Roquefort  Penicillium  and  kept  in  a  room  at  a  temperature  of  about 
60°  F.  At  the  end  of  five  weeks  this  cheese  was  found  to  have  ac- 
quired both  the  texture  and  the  flavor  of  genuine  Roquefort.  There 
seems  to  be  no  doubt  that  it  will  be  possible  to  develop  methods  of 
making  and  ripening  that  will  produce  the  Roquefort  type  of  cheese 
successfully  in  the  United  States.  Details  of  making  and  handling 
will  then  be  offered. 

CHEESES  RELATED  TO  ROQUEFORT. 

Single  studies  have  been  made  from  the  Italian  Gorgonzola,  Eng- 
lish Stilton,  and  Hungarian  Brinse  (Brindze  or  Brimse).  Gorgonzola 
and  Stilton  are  made  from  cow's  milk.  Brinse  is  described  as  made 
from  sheep's  milk,  mixed  sometimes  with  goat's  milk.  These  three 
varieties  of  cheese  are  found  marbled  with  green  Penicillia  in  pure 
cultures,  which  are  unquestionably  one  or  more  strains  of  the  Roque- 


30  FUNGI    IN    CHEESE    RIPENING. 

fort  Penicillium.  In  the  Gorgonzola  and  Stilton  cheeses  examined 
lactic  species  were  the  only  bacteria  found.  Comparison  of  the  flavors 
in  these  cheeses  shows  that  the  differences  lie  in  the  qualities  of  the 
materials  used  in  the  making  and  the  handling  of  the  cheeses  rather 
than  in  the  qualities  attributable  to  ripening  organisms.  It  is  pecul- 
iarly interesting  to  find  the  same  species  of  mold  in  the  interior  of 
ripened  cheese  in  four  countries  so  widely  separated,  where  no  efforts 
at  the  use  of  pure  cultures  are  known  to  be  made.  Experiments  show 
that  in  every  locality  so  far  studied  there  are  many  green  species  of 
Penicillium.  It  is  evident,  then,  that  the  food  material  or  the  condi- 
tions, or  both,  presented  by  these  types  of  cheese  must  exert  a  selective 
influence  upon  the  molds,  which  results  in  the  dominance  of  the  one 
species  so  universally  found.  This  species  has  been  introduced  into 
experimental  cheeses  at  this  station. 

AMERICAN  BRIE  AND  ISIGNY. 

Cheeses  of  the  type  referred  to  in  our  previous  bulletin  as  the  Ameri- 
can Brie  have  been  studied  for  comparison.  This  was  a  collective 
term  suggested  to  cover  cheese  sold  under  various  labels  as  Brie, 
Isigny,  Wiener,  Miniature,  and  others,  designated  commonly  by  the 
retailer  simply  as  Brie.  The  name  "Brie  "  seems  to  be  applied  in  the 
French  dairy  literature  to  a  cheese  which  differs  from  the  Camembert 
in  the  process  of  making,  but  ripened  by  the  same  fungi  and  approxi- 
mately in  the  same  way  as  Camembert.  The  domestic  product  so  far 
as  examined  is  quite  different,  with  the  exception  of  the  output  of  one 
factory,  which  is  conducted  by  imported  cheese  makers.  The  cheese 
met  in  the  eastern  markets  under  these  names  shows  no  trace  of  the 
Camembert  Penicillium.  Numerous  brands  have  been  examined  in 
the  market  and  many  hundreds  of  cheeses  have  been  seen  in  the  cel- 
lars of  two  of  the  largest  cheese  companies.  Oidium  lactis  is  univer- 
sally present  upon  these  cheeses,  but  its  presence  goes  practically  un- 
noticed by  the  makers,  since  it  produces  neither  color  nor  aerial 
mycelium.  All  noticeable  molds  are  washed  or  scraped  from  the  sur- 
face of  the  cheese.  The  washing  produces  exactly  the  best  conditions 
for  the  growth  of  bacteria  and  Oidium.  This  treatment  results  in  a 
cheese  without  a  very  definite  fungous  rind  and  with  a  strong  flavor 
and  smell. 

Cultures  from  this  type  of  cheese  indicate  that  there  is  an  asso- 
ciative action  between  the  Oidium  lactis  and  various  species  of  bacte- 
ria. Several  species  of  Penicillium  occur  as  contaminations  in  these 
cellars  and  sometimes  are  found  upon  the  cheeses  in  the  market. 
Every  effort  is  made  to  eliminate  mold  action  other  than  that  of 
Oidium  lactis,  which  usually  passes  unrecognized.  Cheeses  of  this 
type  usually  bear  rich  growths  of  yeasts,  giving  a  characteristic  greasy 
feeling  to  the  surface.  Exactly  what  parts  these  various  organisms 
play  in  the  production  of  Brie  is  as  yet  undetermined. 


CAMEMBERT    AND    ROQUEFORT.  31 

Single  studies  have  shown  that  Oidium  lactis  is  the  dominant  mold 
upon  the  surface  of  some  brands  of  Limburger,  brick,  and  Port  du 
Salut.  There  is,  then,  good  reason  to  believe  that  this  fungus  is  asso- 
ciated with  nearly  every  type  of  highly  flavored,  ripened  soft  cheese 
met  in  the  American  market. 

MOLDS  BEFEBBED  TO  IN  THIS  PAPEB. 

The  Camembert  and  Roquefort  molds  belong  to  the  hyphomycete 
genus  Penicillium,  which  has  been  characterized  by  one  author — 

Hyphae  broadly  effused,  creeping;  conidiophore  branched  at  the  apex  in  an  irregularly 
verticillate  manner,  producing  brush  or  broom-like  forms;  conidia  in  chains,  hyaline  or 
bright  colored,  spherical  or  elliptical. 

This  genus  of  fungi  contains  a  large  number  of  very  poorly  de- 
scribed forms  which  are  everywhere  abundant  as  the  "green"  or 
"blue"  mold  of  the  household,  the  dairy,  and  the  granary.  They 
form  patches  upon  and  just  under  the  surface  of  the  materials  upon 
which  they  grow.  The  patches  are  composed  of  delicate  threads  of 
mold,  which  are  matted  together,  forming  more  or  less  cottony  sur- 
faces, never  rising  more  than  a  small  fraction  of  an  inch  above  the  sub- 
stratum. At  first  these  areas  are  always  white,  but  in  most  species 
the  ripening  of  a  crop  of  spores  is  indicated  by  the  change  to  a  color 
which  is  usually  some  shade  of  green,  though  this  may  later  give 
place  to  a  brown.  In  a  few  species  other  colors  appear.  These  spores 
(conidia),  or  propagating  bodies,  are  minute  thin-walled  cells  averag- 
ing possibly  one  five-thousandth  of  an  inch  in  diameter,  and  so  light 
that  they  float  freely  in  the  air.  A  breath  upon  the  surface  of  such  a 
colony  carries  away  thousands  of  them,  when  if  held  in  a  proper  posi- 
tion they  may  commonly  be  seen  to  rise  in  a  cloud.  If  the  colony  be 
held  to  the  nose  and  inhaled  they  give  the  sensation  commonly  called 
the  "smell  of  mold."  They  are,  then,  exceedingly  light;  they  are  pro- 
duced in  immense  numbers;  they  are  capable  of  growing  in  almost 
every  conceivable  situation,  upon  anything  which  is  not  definitely  and 
strongly  poisonous.  Some  of  these  spores  are  short  lived,  others  cling 
tenaciously  to  their  power  to  germinate.  Of  the  species,  probably  a 
dozen  common  ones  may  be  expected  in  any  locality,  perhaps  more. 
Our  studies  have  shown  that  they  affect  very  differently  the  sub- 
stances upon  which  they  grow.  It  is,  then,  clearly  necessary  that  by 
thorough  study  of  their  characters  and  habits  we  know  the  forms  we 
are  to  use,  and  just  as  important  that  we  know  how  to  get  rid  and  stay 
rid,  if  it  be  possible,  of  those  we  do  not  want.  The  discussion  of  the  whole 
group  will  be  reserved  for  another  paper.  Here  we  may  describe  in 
simple  terms  the  two  cheese  fungi  we  find  important,  but  it  may  as 
well  be  acknowledged  at  the  outset  that,  with  the  possible  exception 
of  the  Camembert  species,  safe  recognition  of  species  without  technical 
knowledge  and  cultural  study  is  out  of  the  question. 


32 


FUNGI    IN    CHEESE    RIPENING. 


THE    CAMEMBERT    MOLD    (PENICILLIUM    CAMEMBERTl). 

The  spores  of  the  Camembert  mold  grow  rather  slowly  in  compari- 
son with  the  other  molds  of  the  group.  They  first  swell  to  nearly 
double  size,  and  then  produce  fine  threads  or  hyphse  at  from  one  to 
three  points  on  their  surface.  Upon  a  cheese  or  in  laboratory  culture 
the  subsequent  growth  of  these  threads  forms  a  colony  large  enough  to 
be  visible  to  the  naked  eye,  in  ordinary  room  temperature,  in  about  two 
days.  Usually  in  four  or  five  days  the  colony  will  have  become  loosely 
white,  cottony,  about  one-half  inch  or  less  in  diameter,  and  perhaps 

standing  one  -  twentieth  of  an  inch 
above  the  surrounding  surface.  At 
or  about  this  stage  the  center  of  this 
colony  begins  to  turn  a  shade  of  green- 
ish gray,  which  is  characteristic  of 
this  species,  though  one  or  two  other 
forms  produce  colors  closely  resem- 
bling this  shade,  and  difficult  to  dis- 
tinguish from  it  except  to  one  very 
familiar  with  the  colors  in  question. 
This  is  due  to  the  presence  of  ripe 
spores.  Upon  the  cheese  in  the  cellar 
this  color  often  does  not  appear  in  less 
than  a  week  or  even  ten  days.  Micro- 
scopic examination  shows  that  the 
submerged  threads  of  mycelium  of 
such  a  colony  do  not  go  deeper  into  the 
solid  media  than  one-sixteenth  of  an 
inch,  and  that  the  superficial  portion 
of  the  mycelium  spreads  as  fast,  or 
nearly  so,  as  the  part  beneath  the  sur- 
face of  the  substratum.  This  fungus 
grows  and  fruits  for  about  two  weeks — 
in  some  cases  this  may  be  prolonged 
to  three  weeks — and  at  the  end  of 
that  period  no  further  growth  is  to  be  expected  from  the  primary 
colonies,  nor,  if  the  medium  is  undisturbed,  is  there  a  secondary 
growth  from  the  germination  of  the  spores  produced  by  the  first 
colony.  In  case  the  rind  of  the  cheese  is  broken  so  that  a  fresh 
surface  is  presented,  the  spores  will  develop  new  colonies  upon  such 
areas.  A  colony,  then,  produces  a  single  crop  of  spores  and  dies, 
under  ordinary  circumstances,  and  in  undisturbed  cultures  there  is 
usually  no  second  growth  from  the  spores  or  from  the  old  mycelium, 
although  the  contrary  has  been  claimed  for  this  fungus  by  a  recent 
writer  (Maze9).     A  cheese  inoculated  with  this  mold  will  become 


Fig.  1. — Camembert  Penicillium  (P.  ca- 
memberti).  a,  conidiophore  showing  a 
common  type  of  branching  and  the  pro- 
duction of  basidia  and  conidia,  highly 
magnified;  b,  a  common  form  showing 
much  less  branching;  c,  d,  /,  diagrams 
of  large  fructifications  (x  80);  g,  i,  j, 
germinating  conidia. 


CAMEMBKRT    AND    ROQUEFORT.  33 

covered  with  pure  white  cottony  mycelium  in  about  a  week.  The 
color  will  then  begin  to  show  the  gray -green  shade  characteristic 
of  the  species,  which  spreads,  until  at  the  end  of  the  second  week  the 
entire  surface,  if  left  undisturbed,  will  be  colored. 

Persistent  search  has  failed  to  find  a  single  colony  in  America  whose 
presence  can  be  attributed  to  anything  but  Camembert  cheese  im- 
ported from  Europe.  The  mold  may  then  be  regarded  as  a  typical 
dairy  form  which  is  not  well  adapted  to  cosmopolitan  conditions  and 
to  the  struggle  for  existence  on  all  sorts  of  media.  In  fact,  in  the 
course  of  laboratory  practice  involving  thousands  of  cultures,  even  in 
the  laboratories  of  this  station,  this  mold  rarely  appears  as  a  contam- 
ination, although  it  has  been  cultivated  in  quantity  and  used  in  the 
inoculation  of  large  numbers  of  cheeses  in  the  same  building  with  the 
bacteriological  laboratory.  Moreover,  the  spores  are  easily  killed  by 
heat  and  retain  their  vitality  for  only  a  few  weeks  in  ordinary  cultures 
allowed  to  dry  in  the  air  at  room  temperature. 

TECHNICAL    CHARACTERIZATION    OF    THE    CAMEMBERT    MOLD.0 

The  following  technical  characterization  of  Penicillium  camemberti 
(fig.  1)  may  be  offered,  based  upon  studies  made  upon  the  sugar 
gelatin  and  potato  agar  described  in  this  paper: 

Colonies  effused,  white,  slowly  changing  to  gray-green  (glaucous);  surface  of  colony  floc- 
cose,  of  loosely  felted  hyphae  ahout  5  /i  in  diameter;  reverse  of  colony  yellowish  white; 
conidiophores  300  to  800  fi  in  length,  3  to  4  /.i  in  diameter,  septate,  cells  thin-walled,  often 
collapsing  in  age,  arising  as  branches  of  aerial  hyphen1;  fructification  sometimes  175  //  in 
length,  but  usually  much  less,  consisting  commonly  of  one  main  branch  and  one  lateral 
sparingly  branched  to  produce  rather  few  basidia,  which  l)oar  long,  loosely  divergent  chains 
of  conidia.  Basidia  8  to  11  by  2.4  to  3  //;  conidia  at  first  cylindrical,  then  elliptical,  and 
finally  globose  when  ripe,  smooth,  bluish-green  by  transmitted  light,  thin-walled  and  com- 
monly guttulate,  4.5  to  5.5  /<  in  diameter,  swelling  in  germination  to  8  to  10  //.  Germ- 
tubes  one  to  several.  Cells  of  mycelium  about  5  by  20  to  40  // ;  liquefies  sugar  gelatin  only 
under  the  center  of  the  colony.  Changes  blue  litmus  to  red  strongly  at  first,  then  after  four 
to  six  days  begins  to  turn  the  red  back  to  blue  at  the  center  and  continues  outward  concen- 
trically until  all  has  become  blue.  Growing  and  fruiting  period  about  two  weeks.  Fruits 
only  upon  exposed  surfaces  of  the  substrata — never  produces  spores  in  cavities  not  very 
broadly  open.     Habitat,  cheese. 


a  Penicillium  camemberti  (noinen  novum).  This  species  is  unquestionably  the  one 
referred  to  by  Maz6  in  his  recent  papers  as  P.  album  Epstein.  Professor  Maze  was  kind 
enough  to  show  me  the  cultures.  But  the  name  I'-  album  was  already  used  by  Preuss  some 
fifty  years  earlier  for  a  species  of  Penicillium,  hence  by  the  rules  of  nomenclature  should  not 
lx'  used  again  for  a  species  whose  identity  with  P.  album  Preuss  is  not  claimed  by  Epstein. 
Upon  this  ground  Lindau,  in  Rabenhorst's  Kryptogamennora,  has  changed  the  name  of 
Epstein's  fungus  to  /'.  epsteini  Lindau.  and  extracted  from  the  article  written  by  Epstein  a 
brief  and  totally  insufficient  diagnosis.  A  careful  study  of  the  physiological  data  given  by 
Epstein  shows  that  they  dilfer  from  the  data  so  far  found  for  this  species  so  materially  as  t" 
lead  to  the  probability  that  he  was  studying  another  form  entirely.  I  therefore  give  /'. 
album  Epstein  in  the  list  of  possible  synonymy  only,  because  the  name  is  accepted  by  Maze 
for  what  I  know  to  be  this  species. 


34  FUNGI    IN    CHEESE    RIPENING. 

THE    ROQUEFORT    MOLD    (PENICILLIUM    ROQUEFORTl). 

The  spores  of  the  Roquefort  mold  grow  very  rapidly,  often  produc- 
ing new  mycelium  and  ripe  spores  within  thirty-six  hours.  The  colo- 
nies are  white  at  the  very  first,  but  begin  to  become  green  at  the  cen- 
ter within  two  days  in  a  rapidly  growing  colony.  Such  a  colony  may 
become  a  half  inch  in  diameter  in  the  first  two  days.  The  mycelium 
is  mostly  submerged,  but  very  close  to  the  surface,  and  grows  rapidly 
outward  from  the  starting  point  in  a  radial  manner,  which  is  rendered 
prominent  by  certain  of  the  threads  lying  just  under  the  surface  for  the 
most  part,  but  making  loops  into  the  air  by  rising  just  above  the  sub- 
stratum for  a  little  way,  then  reentering  the  medium  again.  This 
gives  a  grayish,  almost  cobwebby  (arachnoid),  appearance  to  the  mar- 
gin of  the  young  colony.  The  rate  of  growth  is  not  uniform  in  the  cir- 
cumference of  such  a  colony,  which  makes  the  border  of  a  colony 
uneven  instead  of  regularly  circular,  as  most  species  appear.  The 
superficial  portion  of  the  Roquefort  mold  is  almost  entirely  composed 
of  the  fruiting  hyphae  or  conidiophores,  the  vast  majority  of  which 
arise  as  branches  of  submerged  hyphae  and  consequently  stand  sepa- 
rately as  short,  unbranched  threads  of  approximately  equal  length, 
which  gives  the  surface  a  velvety  appearance.  They  are  usually  0.2 
or  0.3  mm.  or  less  in  length,  say  one  seventy-fifth  of  an  inch.  Such  a 
colony  spreads  indefinitely  in  the  substratum,  so  that  the  center  will 
be  composed  of  ripe  fruit,  while  the  margin  is  still  actively  growing. 
In  laboratory  culture,  however,  the  development  is  so  rapid  that  the 
entire  surface  is  covered  within  the  first  few  days;  then  growth  ceases. 
The  mycelium  here,  as  in  the  Camembert  mold,  produces  but  a  single 
crop  of  spores,  then  dies.  These  spores  are  a  bright  green  at  first,  but 
in  a  short  time  become  a  dirty-brown  color  in  dry  culture.  The  spores 
of  this  fungus  are  much  more  resistant  than  those  of  the  Camembert 
mold  both  to  heat  and  to  natural  exposures.  They  will  retain  their 
viability  for  months  in  old  cultures  under  the  ordinary  conditions  of 
exposure  in  the  laboratory.  Upon  a  cheese  this  mold  produces  a 
bright  green  area  which  extends  rapidly.  Its  action  can  be  detected 
in  a  few  days  by  the  bitter  taste  of  the  curd  near  to  the  mycelium.  A 
similar  taste  is,  however,  produced  at  least  in  some  measure  by  other 
green  forms,  so  that  it  is  not  diagnostic  except  as  between  this  and  the 
Camembert  species.  A  colony  upon  the  surface  of  a  cheese  becomes 
brown  in  two  or  three  weeks,  but  colonies  growing  in  the  cavities 
which  are  so  characteristic  of  the  center  of  this  type  of  cheese  retain 
their  bright  green  color  for  long  periods. 

This  mold  is  not  limited  to  dairy  products,  but  is  widely  distributed. 
It  has  been  sent  to  the  laboratory  from  the  most  distant  correspond- 
ents. It  has  been  found  in  silage,  and  in  laboratory  cultures  from 
many  substances.  It  has  been  found  to  be  the  green  mold  of  Stilton, 
Gorgonzola,  and  Brinse,  as  well  as  in  certain  tvpes  of  prepared  cheese 


CAMEMBERT    AND    ROQUEFORT. 


35 


purchased  in  the  market.  Once  in  a  laboratory  it  stays  and  seems 
to  get  into  everything.  In  other  words,  this  is  one  of  the  cosmopoli- 
tan and  omnivorous  species  of  the  genus.  One  character  seems  to 
differentiate  this  mold  from  most  of  the  others — that  is,  its  power  of 
growing  into  and  fruiting  normally  within  narrow  cavities,  such  as 
appear  in  cheese.  It  appears  that  this  character  exerts  a  sort  of 
automatic  (perhaps  we  may  call  it  a  truly  "natural")  selection  which 
eliminates  all  other  species  from  the  ripening  processes  of  Roquefort 
and  related  types  of  cheese. 


Pig.  2.— Roquefort  Penicillium  (P.  roqueforti).  a,  part  of  eonidiophore  and  of  bas  of  fructification, 
highly  magnified,  showing  the  production  of  basidia  on  the  sides  as  well  as  at  the  apex  of  the 
basidiophore;  b,  c,  other  types  of  branching;  <l,  young  eonidiophore  just  branching;  e,  /,  basidia 
and  the  formation  of  conidia,  highly  magnified;  g,  h,j,  diagrams  of  types  of  fructification  as  seen 
under  low  power  (x  80);  k,  I,  m,  n,  germination  of  conidia  and  new  conidia  produced  directly  on 
the  first  hyphse. 

TECHNICAL  CHARACTERIZATION  OF  THE  ROQUEFORT  MOLD.** 

A  technical  characterization  is  offered  of  Penicillium  roqueforti 
(fig.  2),  as  follows: 

Colonies  quickly  turning  green,  becoming  a  dirty  brown  in  age,  velvety  strict,  indetermi- 
nately spreading  by  large  main  radiating,  brandling  hypha;,  giving  a  somewbat  uneven  or 

a  Penicillium  roqueforti  (nomen  novum)-  In  offering  a  new  specific  name  for  this  well- 
known  fungus,  the  author  is  perfectly  aware  that  the  mold  is  often  referred  to  in  the  litera- 
ture as  P.  glaucum.  A  careful  study  of  the  literature  fails  to  disclose  a  single  description 
which  indicates  that  this  is  identical  with  the  plant  described  as  P.  glaucum.  As  a  prelimi- 
nary step,  therefore,  to  the  proper  determination  of  the  green  species  of  Penicillium  which 
have  hitherto  l>een  collectively  referred  to  as  /'.  t/laucum,  this  very  distinct  and  easily  rec- 
ognized form  is  named  from  its  universal  occurrence  P.  roqueforti. 


36  FUNGI    IN    CHEESE    RIPENING. 

indefinite  margin,  which  gets  a  white,  fibrous,  almost  spider-web  appearance  from  its  altar- 
nation  of  submerged  parts  of  hyphffi  with  short  prostrate  aerial  loops;  reverse  of  colony  yel- 
lowish white.  Conidiophores  arising  separately  and  in  acropetal  succession  from  the  grow- 
ing parts  of  submerged  hyphte  (comparatively  few  from  aerial  parts,  but  some),  200  to  300  // 
septate  Fructification  90  to  120  //  or  at  times  160  //  by  30  to  60//  at  broadest  place, 
usually  appearing  double  by  the  divergence  of  the  lowest  branch;  branchlets  (basidiophores) 
irregularly  verticillate,  bearing  crowded  verticils  of  appressed  basidia  9  to  11  //  by  2.5// 
with  long  divergent  chains  of  conidia.  Conidia  bluish  green,  cylindrical  to  globose,  smooth, 
rather  finn-walled,  4  to  5  //  in  diameter,  germinating  by  a  straight  tube.  Colonies  do  not 
liquefy  sugar  gelatin,  though  they  soften  it  somewhat.  The  fungus  changes  litmus  from 
fed  to  blue  very  rapidly  and  strongly,  almost  from  the  beginning  of  growth.  Fruiting  period 
short,  but  one  crop  of  spores  upon  the  mycelium.  Cosmopolitan  and  omnivorous,  or  nearly 
so.    Characteristic  of  Roquefort  and  related  types  of  cheese. 

OIDIUM    LACTIS. 

The  mold  (fig.  3)  variously  known  as  Oidium,  or  Oospera,  lactis 
is  another  cosmopolitan  organism.  This  fungus  differs  widely  from 
the  species  previously  described.  Inoculated  into  any  suitable 
medium  it  grows  with  enormous  rapidity.  A  single  spore  (or  oidium) 
may  give  rise  to  several  centimeters  of  mycelium  and  hundreds  of 
spores  in  twenty-four  hours.  It  prefers  very  moist  situations,  since 
almost  the  entire  mycelium  is  developed  below  the  surface  of  the  sub- 
stratum. It  is  therefore  passed  unnoticed  many  times  or  produces 
changes  which  are  attributed  by  the  observer  to  bacteria.  Descrip- 
tion, therefore,  must  depend  upon  microscopic  characters.  The  study 
of  the  border  of  the  young  colony  shows  numerous  vegetative  hyphse 
radiating  outward.  Each  of  these  is  found  to  divide  dichotomously 
(fig.  3,  a,  b),  so  that  the  border  is  a  crowded  series  of  forking  branches. 
In  the  older  parts  of  the  mycelium  a  branch  may  be  produced  at  each 
end  of  eArery  cell,  or  several  at  each  end,  and  these  branch  indefinitely. 
The  fruiting  branches  are  mostly  produced  as  outgrowths  from  the  dis- 
tal ends  of  the  cells.  These  extend  upward  into  the  air  or  remain  en- 
tirely submerged  in  many  cases.  From  the  ends  of  these  outgrowths 
one  to  several  rows  of  oblong  or  cylindrical  cells  begin  to  be  pinched  off. 
If  extending  above  the  surface  this  gives  rise  to  chains  of  delicate  shim- 
mering cells  appearing  as  a  powdery  covering  upon  the  surface,  which 
can  be  seen  with  a  good  lens  to  be  arranged  in  chains.  In  some  strains 
of  Oidium  all  of  these  chains  (and  some  of  the  chains  in  all  strains)  of 
spores  remain  submerged  and  germinate  at  once,  so  that  they  give 
rise  to  unintelligible  mats  of  hyphae.  Oidium  produces  a  very  slight 
acid  reaction  to  litmus  at  first,  then  a  strong  and  continued  alkaline 
reaction.  It  liquefies  sugar  gelatin  under  the  colonies,  but  does  not 
extend  the  area  of  liquefaction  beyond  the  edge  of  the  colony. 
Oidium  always  and  everywhere  tested  has  produced  a  strong  and  very 
characteristic  odor.  Once  familiar  with  this  odor  the  worker  may 
recognize  its  presence  by  its  spores  or  oidia,   which  are  hyaline, 


CAMEMBEKT   AND   ROQUEFORT. 


37 


smooth,  cylindrical,  3.5  to  5  /*  by  6  to  30  M,  varying  with  the  condi- 
tions and  the  substratum  and  perhaps  at  times  exceeding  these  limits. 
These  swell  variously  and  germinate  in  many  ways,  so  that  no  germi- 
nation characters  are  definite.  Upon  some  media  this  mold  may  be 
induced  to  produce  a  large  growth  of  aerial  mycelium,  but  the  limits 
here  denned  will  include  the  variations  to  be  found  upon  the  usual 
culture  media. 

Oidium  lactis  is  described  as  universally  present  on  milk  and  its 
products.  Epstein  even  suggests  that  experiments  upon  milk  and 
cheese  can  not  be  freed  from  its  presence  without  sterilizing.     The 


Fig.  3. — Oidium  lactis.  a,  b,  dichotomous  branching  of  growing  hyptaee;  e,  d,  g,  simple  chains  of  oidia 
breaking  through  substratum  at  (lotted  line  a--//,  dotted  portions  submerged;  e,f,  chains  of  oidia 
from  a  branching  outgrowth  of  a  submerged  cell;  h,  branching  chain  of  oidia;  k,  I,  m,  n,  o,  p,  s, 
types  of  germination  of  oidia  under  varying  conditions;  t,  diagram  of  a  portion  of  a  colony  show- 
ing habit  of  Oidium  lactis  as  seen  in  culture  media. 

same  or  almost  indistinguishable  forms  are  found  upon  decaying  vege- 
tables and  fruits,  which  may  give  reason  for  the  statement  that  the 
odor  produced  by  Oidium  is  that  of  rotten  cabbage.  There  seems  to 
be  good  reason  for  saying  that  all  these  forms  are  but  varieties  or 
strains  of  the  same  species.  Comparison  of  several  of  them  shows 
that  under  uniform  conditions  the  morphology  of  all  these  forms  is 
very  nearly  the  same.  This  is  largely  true  also  of  their  physiological 
effects.  This  mold  has  been  much  studied  and  numerous  papers  dis- 
cuss its  nature  and  physiological  effects  as  well  as  its  relationships. 


38  FUNGI    IN    CHEESE    RIPENING. 

It  will  be  sufficient  to  describe  here  the  fungus  and  to  give  figures  to 
assist  in  its  recognition.  Its  relations  to  the  problems  of  cheese  ripen- 
ing have  already  been  indicated. 

SUMMARY. 
CAMEMBERT    CHEESE. 

The  acidity  of  the  curd  resulting  from  the  action  of  lactic  organ- 
isms reduces  where  it  does  not  entirely  eliminate  the  growth  of  objec- 
tionable bacteria. 

Many  species  of  dairy  fungi  exert  in  the  course  of  their  development 
the  power  of  changing  this  reaction  to  alkaline.  The  Camembert 
Penicillium  and  Oidium  lactis  possess  this  power,  but  not  in  greater 
degree  than  many  other  species. 

Many  species  of  fungi  possess  the  ability  to  change  curd  to  a  greater 
or  less  extent. 

The  breaking  down  of  curd  by  fungi  is  due  in  the  cases  studied  to 
the  production  of  enzymes. 

•  The  texture,  appearance,  and  flavor  of  curd  acted  upon  by  such 
fungi  are  different  for  different  species. 

The  Camembert  Penicillium  (P.  camemherti)  is  the  only  species  so 
far  studied  with  which  the  particular  appearance  and  texture  sought 
in  the  ripened  Camembert  can  be  produced  from  curd  soured  by 
lactic  bacteria  without  producing  any  objectionable  flavor. 

Oidium  lactis  is  always  found  upon  Camembert  cheese  and  so  closely 
associated  with  the  presence  of  the  flavor  as  to  indicate  its  agency  in 
flavor  production,  though  only  circumstantial  proof  of  such  function 
has  been  possible  thus  far.  The  participation  of  bacteria  in  flavor 
production  is  not  excluded  by  these  results. 

Other  species  of  fungi  have  been  shown  to  produce  variations  in 
this  flavor  such  as  have  been  often  found  in  certain  market  cheeses. 
In  this  way  it  is  possible  to  look  for  the  cause  of  differences  in  flavor 
in  contamination  of  the  cultures  upon  the  cheeses.  This  points 
toward  the  use  of  pure  cultures  for  inoculation,  with  the  addition  of 
special  organisms  if  certain  variations  from  what  we  have  regarded  as 
typical  flavor  are  found  to  be  of  value  in  the  market  rather  than 
dependence  upon  accidental  occurrence  of  the  desired  species  in  the 
factory. 

ROQUEFORT    CHEESE. 

In  the  ripening  of  Koquefort  cheese  the  only  organisms  found  neces- 
sary are  lactic  bacteria  and  the  Roquefort  species  of  Penicillium. 

The  Roquefort  Penicillium  has  been  shown  to  possess  the  power  to 
reduce  the  acidity,  to  digest  the  curd,  and  to  produce  the  typical 
flavor. 


CAMEMBERT    AND    ROQUEFORT.  39 

OTHER    VARIETIES    OF   CHEESE. 

The  Roquefort  species  of  Penicillium  is  found  in  the  imported  Stil- 
ton, Gorgonzola,  and  Brinse,  as  well  as  in  Roquefort  cheese. 

Oidium  lactis  alone  of  the  forms  studied  has  been  found  upon  the 
various  brands  of  Limburger,  Brie  (American  type),  Isigny,  and 
related  cheeses  found  in  the  market.  Other  species  incidentally 
occur,  but  not  uniformly,  and  such  occurrence  is  avoided  as  far  as 
possible  by  the  makers. 


BIBLIOGRAPHY. 

(1)  Conn,  Hekbert  William;  Thom,  Charles;  Boswortii,  A.  W.;  Stocking,  W.  A.,  Jr., 

and  Issajeff,  T.  W.  The  Camembert  type  of  soft  cheese  in  the  United  States. 
Bull.  No.  71,  U.  S.  Department  of  Agriculture,  Bureau  of  Animal  Industry. 
Washington,  1905.  Also  published  as  Bull.  No.  35  of  the  Storrs  Agricultural 
Experiment  Station,  Storrs,  Conn.,  Apr.,  1905. 

(2)  Conn,  Herbert  William.     Bacteria  in  milk  and  its  products.     Mus.     306.  pp.   Phila- 

delphia, Blakiston's  Sons  &  Co.,  1903.     See  p.  268. 

(3)  Epstein,  Stanislaus.     Untersuchungen  uber  die  Reifung  von  Weichkasen.     Arch.  f. 

Hyg.,  Bd.  43,  Hft.  1,  pp.  1-20;  Bd.  45,  Hft.  4,  pp.  354-376.  Munich  and  Leipzig, 
1902. 

(4)  Johan-Olsen,  Olav.     Die  bei  der  Kasereifung  wirksamen  Pilze.     Cent.  f.  Bakt.,  Abt. 

2.  Bd.  4,  No.  5,  pp.  162-169.     Jena,  March  5,  1898. 

(5)  Constantin,  J.,  and  Ray,  J.     Sur  les  champignons  du  fromage  de  Brie.     Compt.  rend. 

Soc.  de  biol.,  Paris,  se>.  10,  t.  5,  No.  16,  pp.  504-507.     Paris,  May  13,  1898. 

(6)  Roger,  Georges.     [Article  in]  Revue  hebdomadaire,  v.  7,  p.  334.     Paris. 

(7)  Margaret,  pseudonym.     The  practice  of  cheesemaking  at  home  and  abroad.     The 

Creamery  Journal,  v.  1,  No.  11,  pp.  313-315.     London,  July  20,  1905. 
<8)  Epstein,  Stanislaus.     See  Citation  3,  above,  p.  373. 

(9)  Maze,  P.     Les  microbes  dans  l'industrie  fromagere.     Ann.  de  l'lnst.  Past.,  ann.  19, 

No.  6,  pp.  378-403,  June  25;  No.  8,  pp.  481-493,  August  25.     Paris,  1905. 

(10)  Smith,  Erwtn  F.,and  Swingle,  Dean  B.     The  dry  rot  of  potatoes,  due  to  Fusarium 

oxysporum.     Bull.  No.  55,  U.  S.  Department   of  Agriculture,  Bureau  of   Plant 

Industry.     Washington,  February  16,  1904. 
Lang,  M.,  and    Freudenreich,  Eduard  von.     Uber  Oidium  lactis.     Landwirthschaftl. 

Jahrbuch  der  Schweiz,  Bd.  7,  pp.  229-237.     Bern,  1893. 
Makpmann,  G.     Beitrage  zur  Kaseflora.         Ztschr.  f.  angewandte  Mikroskopie,  Bd.  2,  Hft. 

3,  pp.  68-79.     Berlin,  June.  1896. 

Teichert,  Kurt.  Beitrage  zur  Biologic  einiger  in  Molkereiproduction  vorkommenden 
Schimmelpilzen.  Milch-Xeitung,  v.  32,  No.  50,  pp.  786-787.  Bremen,  December 
12, 1903. 

Thom,  Charles.  Some  suggestions  from  the  study  of  dairy  fungi.  Jrn.  of  Mycology,  v.  2, 
NO.  77,  pp.  117-124.     Columbus,  Ohio,  May,  1905. 

o 


R^nm  .kV  '    °*  ^fl6^,  CA  90024-iaaa 
fr«™  ,h[V"a«erial  to  the  library      *" 
J^L!!^J!r^orrowed 


I 


ND    ERADICATION    OF    CONTAGIOUS   DISEASES. 


Inspectors  in  charge  of  districts 


1 


room  328  Livi 


^_                                            **20  Qiimey  Building,  Fargo,  N.  Dak.— Dr.  R.  II.  Treaey. 

Deme.,                               urge  of  eradication  of  KansasCity,  Kans.— Albert  Dean',] 

lies  of  sheep  and  cattle  in  the  Wesfe  Stock  exchange; 

Albuquerque.  N.  Mex.— Dr.  Louis  Metsker,  room  Salt  LakeClty;  Utah.— Dr.  OeorgeS.  Hiotox  room 

22  X.  T.  Annijo  Building.  21  Eagle  block. 
Denver,  Colo.— Dr.  Lowell  Clarke,  room  320  Quinev 

Building. 

INSPECTION    OF   LIVE   STOCK   FOR   EXPORT. 

Inspectors  in  charge. 


Baltimore,  Md.— Dr.  11.  A.  Hedrkk,  215  St.  Paul 

street. 
New  York.  N.  Y.— Dr.  W.  H.  Rose,  IS  Broadway. 
Norfolk,  Va.— Dr.  G.  C.  Faville,  P.  <>.  bos 


Philadelphia,  Pa.— Dr.  C.  A.  Schauflcr.  134  South 
Second  si  h 

Portland,  Mo.  — Dr.  F.  \V.  Huntington,  U.  S.  cus- 
toms office,  Grand  Trunk  R.  R.  whan. 


INSPECTION    AND   QUARANTINE   OF    IMPORTED   ANIMALS. 

(Quarantine  stations. 

Athenia,  N.  J.  (for  the  port  of  New  York).— Dr.  I  Littleton,  Mass.    (for  the  port  of  Boston).— Dr 
George  \\ .  Pope,  superintendent.  ,j.  F.  Ryder,  inspector  in  charge,  i-n  Milk  street, 

Balethorp,  Md.  (for  the  port  of  Baltimore) .— Wll-  !       Boston,  Mass. 
liam  II.  Wade,  superintendent. 

Inspectors  on  the  Canadian  border. 


Calais,  Me.— Dr.  II.  T.  Potter. 

Carthage,  N.  Y.—  Dr.  W.  S.  Corlis. 

Detroit.  Mich.— Dr.  L.  K.  Green,  care  Hammond, 

Standish  &  Co. 
Fort  Fairfield,  Me.    Dr.  F.  M.  Perry. 
Malone,  N.  Y.— Dr.  II.  I).  Mavne. 
Newport,  Vt— Dr.  G.  \V.  Ward. 


Ogdensbnrg,  N.  Y.— Dr.  Charles  Cowie. 

Onmo,  Me.     Dr.  F.  L.  Hi; 

Port     Huron,    Mich.  — Dr.    David    Cumming.    912 

Lapegr  avenue. 
St.  Albans,  VV- Dr.  C.  L.  Morin. 
Sault  Ste.  Marie,  Mich.— Dr.  J.  F,  Deadman. 


Inspectors  on  the  Mexican  border. 


El  Paso,  Tex.— Dr.  Thomas  A.  Brav. 

San  Antonio,  Tex.— Dr.  .Joseph  \V.  Parker. 


San  Diego,  Cal.— Dr.  Robert  Darling,  care  Charles 

S.  Hardy. 


VETERINARY    INSPECTORS  STATIONED   ABROAD. 

Dr.  \Y.  H.\Y ray, 3tStreathaii:  Hill.  London,  S.  U\.  Dr.  James  Johnston,  care  t".  S.  consulate   Liver- 

Kuglund. in  charge  for  Great  Britain  and  Ireland.  pool,  England. 

Dr.  T.  A.  Geddes,  care    V.  S.  consulate,  London,  l^r.  V.  A.  Norgaard,  Honolulu,  Hawaii 

England. 


DAIRY   INSPECTORS. 


M.  W.  Lang,  room  23  Marine  Building,  C: 

Robert  MeAdam,  room  23  Marine  Building,  Chi- 
lli. 
E.  A.  McDonald  58  and  59  Downs  block,  Seattle, 
Wash. 


B.  P.  Van  Valkenburgh,  168 Chambers  street.  Now 

York,  V  Y. 
Levi  Wells,  Spring  Hill,  Pa. 
G.  M.  Vv'hitaker  P.  O.  box  1^32,  Boston,  Mass. 


OTUOJIT8 


